Cross-species-specific single domain bispecific single chain antibody

ABSTRACT

The present invention relates to a bispecific single chain antibody molecule comprising a first binding domain consisting of one antibody variable domain capable of binding to an epitope of the human and non-chimpanzee primate CD3 epsilon chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs. 2, 4, 6, and 8, and a second binding domain capable of binding to an epitope of a human and a non-chimpanzee primate tumor target antigen. The invention further relates to a bispecific single chain antibody molecule comprising a first binding domain capable of binding to an epitope of human and non-chimpanzee primate CD3ε (epsilon) chain, wherein the epitope is part of an amino acid sequence comprised in the group consisting of SEQ ID NOs. 2, 4, 6, and 8, and a second binding domain consisting of one antibody variable domain capable of binding to an epitope of a human and a non-chimpanzee primate tumor target antigen. The invention also provides nucleic acids encoding said bispecific single chain antibody molecule as well as vectors and host cells and a process for its production. The invention further relates to pharmaceutical compositions comprising said bispecific single chain antibody molecule and medical uses of said bispecific single chain antibody molecule.

The present invention relates to a bispecific single chain antibodymolecule comprising a first binding domain consisting of one antibodyvariable domain capable of binding to an epitope of the human andnon-chimpanzee primate CD3 epsilon chain, wherein the epitope is part ofan amino acid sequence comprised in the group consisting of SEQ ID NOs.2, 4, 6, and 8, and a second binding domain capable of binding to anepitope of a human and a non-chimpanzee primate tumor target antigen.The invention further relates to a bispecific single chain antibodymolecule comprising a first binding domain capable of binding to anepitope of human and non-chimpanzee primate CD3ε (epsilon) chain,wherein the epitope is part of an amino acid sequence comprised in thegroup consisting of SEQ ID NOs. 2, 4, 6, and 8, and a second bindingdomain consisting of one antibody variable domain capable of binding toan epitope of a human and a non-chimpanzee primate tumor target antigen.The invention also provides nucleic acids encoding said bispecificsingle chain antibody molecule as well as vectors and host cells and aprocess for its production. The invention further relates topharmaceutical compositions comprising said bispecific single chainantibody molecule and medical uses of said bispecific single chainantibody molecule.

T cell recognition is mediated by clonotypically distributed alpha betaand gamma delta T cell receptors (TcR) that interact with thepeptide-loaded molecules of the peptide MHC (pMHC) (Davis & Bjorkman,Nature 334 (1988), 395-402). The antigen-specific chains of the TcR donot possess signalling domains but instead are coupled to the conservedmultisubunit signalling apparatus CD3 (Call, Cell 111 (2002), 967-979,Alarcon, Immunol. Rev. 191 (2003), 38-46, Malissen Immunol. Rev. 191(2003), 7-27). The mechanism by which TcR ligation is directlycommunicated to the signalling apparatus remains a fundamental questionin T cell biology (Alarcon, loc. cit.; Davis, Cell 110 (2002), 285-287).It seems clear that sustained T cell responses involve coreceptorengagement, TcR oligomerization, and a higher order arrangement ofTcR-pMHC complexes in the immunological synapse (Davis & van der Merwe,Curr. Biol. 11 (2001), R289-R291, Davis, Nat. Immunol. 4 (2003),217-224). However very early TcR signalling occurs in the absence ofthese events and may involve a ligand-induced conformational change inCD3 epsilon (Alarcon, loc. cit., Davis (2002), loc. cit., Gil, J. Biol.Chem. 276 (2001), 11174-11179, Gil, Cell 109 (2002), 901-912). Theepsilon, gamma, delta and zeta subunits of the signalling complexassociate with each other to form a CD3 epsilon-gamma heterodimer, a CD3epsilon-delta heterodimer, and a CD3 zeta-zeta homodimer (Call, loc.cit.). Various studies have revealed that the CD3 molecules areimportant for the proper cell surface expression of the alpha beta TcRand normal T cell development (Berkhout, J. Biol. Chem. 263 (1988),8528-8536, Wang, J. Exp. Med. 188 (1998), 1375-1380, Kappes, Curr. Opin.Immunol. 7 (1995), 441-447). The solution structure of the ectodomainfragments of the mouse CD3 epsilon gamma heterodimer showed that theepsilon gamma subunits are both C2-set Ig domains that interact witheach other to form an unusual side-to-side dimer configuration (Sun,Cell 105 (2001), 913-923). Although the cysteine-rich stalk appears toplay an important role in driving CD3 dimerization (Su, loc. cit.,Borroto, J. Biol. Chem. 273 (1998), 12807-12816), interaction by meansof the extracellular domains of CD3 epsilon and CD3 gamma is sufficientfor assembly of these proteins with TcR beta (Manolios, Eur. J. Immunol.24 (1994), 84-92, Manolios & Li, Immunol. Cell Biol. 73 (1995),532-536). Although still controversial, the dominant stoichiometry ofthe TcR most likely comprises one alpha beta TcR, one CD3 epsilon gammaheterodimer, one CD3 epsilon delta heterodimer and one CD3 zeta zetahomodimer (Call, loc. cit.). Given the central role of the human CD3epsilon gamma heterodimer in the immune response, the crystal structureof this complex bound to the therapeutic antibody OKT3 has recently beenelucidated (Kjer-Nielsen, PNAS 101, (2004), 7675-7680).

A number of therapeutic strategies modulate T cell immunity by targetingTcR signalling, particularly the anti-human CD3 monoclonal antibodies(mAbs) that are widely used clinically in immunosuppressive regimes. TheCD3-specific mouse mAb OKT3 was the first mAb licensed for use in humans(Sgro, Toxicology 105 (1995), 23-29) and is widely used clinically as animmunosuppressive agent in transplantation (Chatenoud, Clin. Transplant7 (1993), 422-430, Chatenoud, Nat. Rev. Immunol. 3 (2003), 123-132,Kumar, Transplant. Proc. 30 (1998), 1351-1352), type 1 diabetes(Chatenoud (2003), loc. cit.), and psoriasis (Utset, J. Rheumatol. 29(2002), 1907-1913). Moreover, anti-CD3 mAbs can induce partial T cellsignalling and clonal anergy (Smith, J. Exp. Med. 185 (1997),1413-1422). OKT3 has been described in the literature as a potent T cellmitogen (Van Wauve, J. Immunol. 124 (1980), 2708-18) as well as a potentT cell killer (Wong, Transplantation 50 (1990), 683-9). OKT3 exhibitsboth of these activities in a time-dependent fashion; following earlyactivation of T cells leading to cytokine release, upon furtheradministration OKT3 later blocks all known T cell functions. It is dueto this later blocking of T cell function that OKT3 has found such wideapplication as an immunosuppressant in therapy regimens for reduction oreven abolition of allograft tissue rejection.

OKT3 reverses allograft tissue rejection most probably by blocking thefunction of all T cells, which play a major role in acute rejection.OKT3 reacts with and blocks the function of the CD3 complex in themembrane of human T cells, which is associated with the antigenrecognition structure of T cells (TCR) and is essential for signaltransduction. Which subunit of the TCR/CD3 is bound by OKT3 has been thesubject of multiple studies. Though some evidence has pointed to aspecificity of OKT3 for the epsilon-subunit of the TCR/CD3 complex(Tunnacliffe, Int. Immunol. 1 (1989), 546-50; Kjer-Nielsen, PNAS 101,(2004), 7675-7680). Further evidence has shown that OKT3 binding of theTCR/CD3 complex requires other subunits of this complex to be present(Salmeron, J. Immunol. 147 (1991), 3047-52).

Other well known antibodies specific for the CD3 molecule are listed inTunnacliffe, Int. Immunol. 1 (1989), 546-50. As indicated above, suchCD3 specific antibodies are able to induce various T cell responses suchas lymphokine production (Von Wussow, J. Immunol. 127 (1981), 1197;Palacious, J. Immunol. 128 (1982), 337), proliferation (Van Wauve, J.Immunol. 124 (1980), 2708-18) and suppressor-T cell induction (Kunicka,in “Lymphocyte Typing II” 1 (1986), 223). That is, depending on theexperimental conditions, CD3 specific monoclonal antibody can eitherinhibit or induce cytotoxicity (Leewenberg, J. Immunol. 134 (1985),3770; Phillips, J. Immunol. 136 (1986) 1579; Platsoucas, Proc. Natl.Acad. Sci. USA 78 (1981), 4500; Itoh, Cell. Immunol. 108 (1987), 283-96;Mentzer, J. Immunol. 135 (1985), 34; Landegren, J. Exp. Med. 155 (1982),1579; Choi (2001), Eur. J. Immunol. 31, 94-106; Xu (2000), Cell Immunol.200, 16-26; Kimball (1995), Transpl. Immunol. 3, 212-221). Although manyof the CD3 antibodies described in the art have been reported torecognize the CD3 epsilon subunit of the CD3 complex, most of them bindin fact to conformational epitopes and, thus, only recognize CD3 epsilonin the native context of the TCR. Conformational epitopes arecharacterized by the presence of two or more discrete amino acidresidues which are separated in the primary sequence, but come togetheron the surface of the molecule when the polypeptide folds into thenative protein/antigen (Sela, (1969) Science 166, 1365 and Laver, (1990)Cell 61, 553-6). The conformational epitopes bound by CD3 epsilonantibodies described in the art may be separated in two groups. In themajor group, said epitopes are being formed by two CD3 subunits, e.g. ofthe CD3 epsilon chain and the CD3 gamma or CD3 delta chain. For example,it has been found in several studies that the most widely used CD3epsilon monoclonal antibodies OKT3, WT31, UCHT1, 7D6 and Leu-4 did notbind to cells singly transfected with the CD3-epsilon chain. However,these antibodies stained cells doubly transfected with a combination ofCD3 epsilon plus either CD3 gamma or CD3 delta (Tunnacliffe, loc. cit.;Law, Int. Immunol. 14 (2002), 389-400; Salmeron, J. Immunol. 147 (1991),3047-52; Coulie, Eur. J. Immunol. 21 (1991), 1703-9). In a secondsmaller group, the conformational epitope is being formed within the CD3epsilon subunit itself. A member of this group is for instance mAb APA1/1 which has been raised against denatured CD3 epsilon (Risueno, Blood106 (2005), 601-8). Taken together, most of the CD3 epsilon antibodiesdescribed in the art recognize conformational epitopes located on two ormore subunits of CD3. The discrete amino acid residues forming thethree-dimensional structure of these epitopes may hereby be locatedeither on the CD3 epsilon subunit itself or on the CD3 epsilon subunitand other CD3 subunits such as CD3 gamma or CD3 delta.

Another problem with respect to CD3 antibodies is that many CD3antibodies have been found to be species-specific. Anti-CD3 monoclonalantibodies—as holds true generally for any other monoclonalantibodies—function by way of highly specific recognition of theirtarget molecules. They recognize only a single site, or epitope, ontheir target CD3 molecule. For example, one of the most widely used andbest characterized monoclonal antibodies specific for the CD3 complex isOKT-3. This antibody reacts with chimpanzee CD3 but not with the CD3homolog of other primates, such as macaques, or with dog CD3 (Sanduskyet al., J. Med. Primatol. 15 (1986), 441-451). Similarly, WO2005/118635or WO2007/033230 describe human monoclonal CD3 epsilon antibodies whichreact with human CD3 epsilon but not with CD3 epsilon of mouse, rat,rabbit, or non-chimpanzee primates, such as rhesus monkey, cynomolgusmonkey or baboon monkey. The anti-CD3 monoclonal antibody UCHT-1 is alsoreactive with CD3 from chimpanzee but not with CD3 from macaques (owndata). On the other hand, there are also examples of monoclonalantibodies, which recognize macaque antigens, but not their humancounterparts. One example of this group is monoclonal antibody FN-18directed to CD3 from macaques (Uda et al., J. Med. Primatol. 30 (2001),141-147). Interestingly, it has been found that peripheral lymphocytesfrom about 12% of cynomolgus monkeys lacked reactivity with anti-rhesusmonkey CD3 monoclonal antibody (FN-18) due to a polymorphism of the CD3antigen in macaques. Uda et al. described a substitution of two aminoacids in the CD3 sequence of cynomolgus monkeys, which are not reactivewith FN-18 antibodies, as compared to CD3 derived from animals, whichare reactive with FN-18 antibodies (Uda et al., J Med Primatol. 32(2003), 105-10; Uda et al., J Med Primatol. 33 (2004), 34-7).

The discriminatory ability, i.e. the species specificity, inherent notonly to CD3 monoclonal antibodies (and fragments thereof), but tomonoclonal antibodies in general, is a significant impediment to theirdevelopment as therapeutic agents for the treatment of human diseases.In order to obtain market approval any new candidate medication mustpass through rigorous testing. This testing can be subdivided intopreclinical and clinical phases: Whereas the latter—further subdividedinto the generally known clinical phases I, II and III—is performed inhuman patients, the former is performed in animals. The aim ofpre-clinical testing is to prove that the drug candidate has the desiredactivity and most importantly is safe. Only when the safety in animalsand possible effectiveness of the drug candidate has been established inpreclinical testing this drug candidate will be approved for clinicaltesting in humans by the respective regulatory authority. Drugcandidates can be tested for safety in animals in the following threeways, (i) in a relevant species, i.e. a species where the drugcandidates can recognize the ortholog antigens, (ii) in a transgenicanimal containing the human antigens and (iii) by use of a surrogate forthe drug candidate that can bind the ortholog antigens present in theanimal. Limitations of transgenic animals are that this technology istypically limited to rodents. Between rodents and man there aresignificant differences in the physiology and the safety results cannotbe easily extrapolated to humans. The limitations of a surrogate for thedrug candidate are the different composition of matter compared to theactual drug candidate and often the animals used are rodents with thelimitation as discussed above. Therefore, preclinical data generated inrodents are of limited predictive power with respect to the drugcandidate. The approach of choice for safety testing is the use of arelevant species, preferably a lower primate. The limitation now ofmonoclonal antibodies suitable for therapeutic intervention in mandescribed in the art is that the relevant species are higher primates,in particular chimpanzees. Chimpanzees are considered as endangeredspecies and due to their human-like nature, the use of such animals fordrug safety testing has been banned in Europe and is highly restrictedelsewhere. CD3 has also been successfully used as a target forbispecific single chain antibodies in order to redirect cytotoxic Tcells to pathological cells, resulting in the depletion of the diseasedcells from the respective organism (WO 99/54440; WO 04/106380). Forexample, Bargou et al. (Science 321 (2008): 974-7) have recentlyreported on the clinical activity of a CD19×CD3 bispecific antibodyconstruct called blinatumomab, which has the potential to engage allcytotoxic T cells in human patients for lysis of cancer cells. Doses aslow as 0.005 milligrams per square meter per day in non-Hodgkin'slymphoma patients led to an elimination of target cells in blood.Partial and complete tumor regressions were first observed at a doselevel of 0.015 milligrams, and all seven patients treated at a doselevel of 0.06 milligrams experienced a tumor regression. Blinatumomabalso led to clearance of tumor cells from bone marrow and liver. Thoughthis study established clincical proof of concept for the therapeuticpotency of the bispecific single chain antibody format in treatingblood-cell derived cancer, there is still need for successful conceptsfor therapies of other cancer types.

In the event that a bispecific antibody is intended for therapeutic use,it is desirable to produce high amounts of this antibody solubly and inthe desired functional form. The production of functionally activeantibody becomes especially critical when producing bispecificantibodies of which one portion is able to activate and recruit thecytotoxic potential of human immune effector cells. For example, aproduced antibody devoid of functional activity will not lead to thedesired activation of human immune effector cells, while a bispecificantibody which is functionally active, albeit not in the desired manner,as for example may be the case when the bispecific antibody is producedin a heterogeneous form containing multiple isomers, may activate andrecruit the cytotoxic potential of human immune effector cells inunforeseeable and/or unintended manners.

One example of the sort of unintended activation mentioned above is thepossibility of activation of human immune effector cells to exert aneffect on other human immune effector cells instead of on a target cellintended for destruction. This type of immune effector cell fratricidemay jeopardize the effectiveness of a regimen of therapy depending onthe activity of human immune effector cells.

However, reliable production of large amounts of functional single chainantibody, especially large amounts of functional bispecific single chainantibody, from prokaryotic expression systems such as E. coli is oftenlimited, necessitating costly optimization (Baneyx 1999. Curr Op inBiotechnol 10, 411-21).

In summary, bispecific antibody constructs can be of great therapeuticuse in redirecting the powerful potential of the body's own immunesystem to achieve the destruction of diseased cells. By the same token,however, the activation of such a powerful means of eradicating orneutralizing unwanted cells requires that this power be controlled asprecisely as possible so that the cytotoxic potential of the immunesystem is recruited and applied only in the direction intended and noother.

Clearly, when one specific binding arm of a bispecific single chainantibody is to recruit the activity of a human immune effector cell, forexample a cytotoxic T cell, there exists an especially heightened and,as yet, unmet need for bispecific single chain antibodies which overcomelimitations as described above.

The present invention provides a bispecific single chain antibodymolecule comprising a first binding domain consisting of one antibodyvariable domain capable of binding to an epitope of the human andnon-chimpanzee primate CD3ε (epsilon) chain, wherein the epitope is partof an amino acid sequence comprised in the group consisting of SEQ IDNOs. 2, 4, 6, and 8, and a second binding domain capable of binding toan epitope of a human and a non-chimpanzee primate tumor target antigen.

Furthermore, the present invention relates to a bispecific single chainantibody molecule comprising a first binding domain capable of bindingto an epitope of human and non-chimpanzee primate CD3ε (epsilon) chain,wherein the epitope is part of an amino acid sequence comprised in thegroup consisting of SEQ ID NOs. 2, 4, 6, and 8, and a second bindingdomain consisting of one antibody variable domain capable of binding toan epitope of a human and a non-chimpanzee primate tumor target antigen.

According to another embodiment of the invention, the first bindingdomain of the bispecific antibody comprises one antibody variabledomain, preferably a VHH domain.

According to one embodiment of the invention, the first binding domainof the bispecific antibody comprises two antibody variable domains,preferably a scFv, i.e. VH-VL or VL-VH.

According to another embodiment of the invention, the second bindingdomain of the bispecific antibody comprises one antibody variabledomain, preferably a VHH domain.

According to one embodiment of the invention, the second binding domainof the bispecific antibody comprises two antibody variable domains,preferably a scFv, i.e. VH-VL or VL-VH.

In its minimal form, the total number of antibody variable regions inthe bispecific antibody according to the invention is thus only two. Forexample, such an antibody could comprise two VH or two VHH domains.Here, not two variable domains VH and VL (a scFv) but rather only onevariable domain is necessary to specifically bind to each antigen ofinterest. The bispecific antibody of the invention is thus approximatelyhalf the size of conventional bispecific single chain antibodiescontaining four antibody variable domains.

The greater simplicity in molecular design of the bispecific antibody ofthe invention correlates to greater possible simplicity in the hostexpression system used for its production in functionally active form.As such, the small size of the inventive bispecific antibody opens upavenues of production hitherto closed to conventional bispecific singlechain antibodies with four antibody variable domains. For example, thebispecific antibody of the invention may be easily produced inconventional, well understood and cheap bacterial expression systemssuch as E. coli in amounts which are desired for therapeuticapplications. Moreover, the bispecific antibody of the invention is morestable than conventional antibodies.

Though T cell-engaging bispecific single chain antibodies described inthe art have great therapeutic potential for the treatment of malignantdiseases, most of these bispecific molecules are limited in that theyare species specific and recognize only human antigen, and—due togenetic similarity—likely the chimpanzee counterpart. The advantage ofthe present invention is the provision of a bispecific single chainantibody comprising a binding domain exhibiting cross-speciesspecificity to human and non-chimpanzee primate of the CD3 epsilonchain.

In the present invention, an N-terminal 1-27 amino acid residuepolypeptide fragment of the extracellular domain of CD3 epsilon wassurprisingly identified which—in contrast to all other known epitopes ofCD3 epsilon described in the art—maintains its three-dimensionalstructural integrity when taken out of its native environment in the CD3complex (and optionally fused to a heterologous amino acid sequence suchas EpCAM or an immunoglobulin Fc part).

The present invention, therefore, provides for a bispecific single chainantibody molecule comprising a first binding domain capable of bindingto an epitope of an N-terminal 1-27 amino acid residue polypeptidefragment of the extracellular domain of CD3 epsilon (which CD3 epsilonis, for example, taken out of its native environment and/or comprised by(presented on the surface of) a T-cell) of human and at least onenon-chimpanzee primate CD3 epsilon chain, wherein the epitope is part ofan amino acid sequence comprised in the group consisting of SEQ ID NOs.2, 4, 6, and 8; and a second binding domain capable of binding toprostate-specific membrane antigen (PSMA). Preferred non-chimpanzeeprimates are mentioned herein elsewhere. At least one (or a selectionthereof or all) primate(s) selected from Callithrix jacchus; Saguinusoedipus, Saimiri sciureus, and Macaca fascicularis (either SEQ ID 427 or428 or both), is (are) particularly preferred. Macaca mulatta, alsoknown as Rhesus Monkey is also envisaged as another preferred primate.It is thus envisaged that antibodies of the invention bind to (arecapable of binding to) the context independent epitope of an N-terminal1-27 amino acid residue polypeptide fragment of the extracellular domainof CD3 epsilon of human and Callithrix jacchus, Saguinus oedipus,Saimiri sciureus, and Macaca fascicularis (either SEQ ID 427 or 428 orboth), and optionally also to Macaca mulatta. A bispecific single chainantibody molecule comprising a first binding domain as defined hereincan be obtained (is obtainable by) or can be manufactured in accordancewith the protocol set out in the appended Examples (in particularExample 2). To this end, it is envisaged to (a) immunize mice with anN-terminal 1-27 amino acid residue polypeptide fragment of theextracellular domain of CD3 epsilon of human and/or Saimiri sciureus;(b) generation of an immune murine antibody scFv library; (c)identification of CD3 epsilon specific binders by testing the capabilityto bind to at least SEQ ID NOs. 2, 4, 6, and 8.

The context-independence of the CD3 epitope provided in this inventioncorresponds to the first 27 N-terminal amino acids of CD3 epsilon orfunctional fragments of this 27 amino acid stretch. The phrase“context-independent,” as used herein in relation to the CD3 epitopemeans that binding of the herein described inventive bindingmolecules/antibody molecules does not lead to a change or modificationof the conformation, sequence, or structure surrounding the antigenicdeterminant or epitope. In contrast, the CD3 epitope recognized by aconventional CD3 binding molecule (e.g. as disclosed in WO 99/54440 orWO 04/106380) is localized on the CD3 epsilon chain C-terminally to theN-terminal 1-27 amino acids of the context-independent epitope, where itonly takes the correct conformation if it is embedded within the rest ofthe epsilon chain and held in the right sterical position byheterodimerization of the epsilon chain with either the CD3 gamma ordelta chain. Anti-CD3 binding molecules as part of a bispecific singlechain antibody molecule as provided herein and generated (and directed)against a context-independent CD3 epitope provide for a surprisingclinical improvement with regard to T cell redistribution and, thus, amore favourable safety profile. Without being bound by theory, since theCD3 epitope is context-independent, forming an autonomous selfsufficientsubdomain without much influence on the rest of the CD3 complex, the CD3binding molecules provided herein induce less allosteric changes in CD3conformation than the conventional CD3 binding molecules, whichrecognize context-dependent CD3 epitopes (e.g. as disclosed in WO99/54440 or WO 04/106380).

The context-independence of the CD3 epitope which is recognized by theCD3 binding domain of the bispecific single chain antibody of theinvention is associated with less or no T cell redistribution (T cellredistribution equates with an initial episode of drop and subsequentrecovery of absolute T cell counts) during the starting phase oftreatment with said bispecific single chain antibody of the invention.This results in a better safety profile of the bispecific single chainantibody of the invention compared to conventional CD3 binding moleculesknown in the art, which recognize context-dependent CD3 epitopes.Particularly, because T cell redistribution during the starting phase oftreatment with CD3 binding molecules is a major risk factor for adverseevents, like CNS adverse events, the bispecific single chain antibody ofthe invention has a substantial safety advantage over the CD3 bindingmolecules known in the art by recognizing a context-independent ratherthan a context-dependent CD3 epitope. Patients with such CNS adverseevents related to T cell redistribution during the starting phase oftreatment with conventional CD3 binding molecules usually suffer fromconfusion and disorientation, in some cases also from urinaryincontinence. Confusion is a change in mental status in which thepatient is not able to think with his or her usual level of clarity. Thepatient usually has difficulties to concentrate and thinking is not onlyblurred and unclear but often significantly slowed down. Patients withCNS adverse events related to T cell redistribution during the startingphase of treatment with conventional CD3 binding molecules may alsosuffer from loss of memory. Frequently, the confusion leads to the lossof ability to recognize people, places, time or dates. Feelings ofdisorientation are common in confusion, and the decision-making abilityis impaired. CNS adverse events related to T cell redistribution duringthe starting phase of treatment with conventional CD3 binding moleculesmay further comprise blurred speech and/or word finding difficulties.This disorder may impair both, the expression and understanding oflanguage as well as reading and writing. Besides urinary incontinence,vertigo and dizziness may also accompany CNS adverse events related to Tcell redistribution during the starting phase of treatment withconventional CD3 binding molecules in some patients.

The maintenance of the three-dimensional structure within the mentioned27 amino acid N-terminal polypeptide fragment of CD3 epsilon can be usedfor the generation of, preferably human, binding domains which arecapable of binding to the N-terminal CD3 epsilon polypeptide fragment invitro and to the native (CD3 epsilon subunit of the) CD3 complex on Tcells in vivo with the same binding affinity. These data stronglyindicate that the N-terminal fragment as described herein forms atertiary conformation, which is similar to its structure normallyexisting in vivo. A very sensitive test for the importance of thestructural integrity of the amino acids 1-27 of the N-terminalpolypeptide fragment of CD3 epsilon was performed. Individual aminoacids of amino acids 1-27 of the N-terminal polypeptide fragment of CD3epsilon were changed to alanine (alanine scanning) to test thesensitivity of the amino acids 1-27 of the N-terminal polypeptidefragment of CD3 epsilon for minor disruptions. The CD3 binding domainsas part of the bispecific single chain antibody of the invention wereused to test for binding to the alanine-mutants of amino acids 1-27 ofthe N-terminal polypeptide fragment of CD3 epsilon (see appended Example5). Individual exchanges of the first five amino acid residues at thevery N-terminal end of the fragment and two of the amino acids atpositions 23 and 25 of the amino acids 1-27 of the N-terminalpolypeptide fragment of CD3 epsilon were critical for binding of theantibody molecules. The substitution of amino acid residues in theregion of position 1-5 comprising the residues Q (Glutamine at position1), D (Aspartic acid at position 2), G (Glycine at position 3), N(Asparagine at position 4), and E (Glutamic acid at position 5) toAlanine abolished binding of the single domain bispecific single chainantibody of the invention to said fragment. While, for at least some ofthe single domain bispecific single chain antibody of the invention, twoamino acid residues at the C-terminus of the mentioned fragment T(Threonine at position 23) and I (Isoleucine at position 25) reduced thebinding energy to the single domain bispecific single chain antibody ofthe invention.

Unexpectedly, it has been found that the thus isolated bispecific singlechain antibody of the invention not only recognizes the human N-terminalfragment of CD3 epsilon, but also the corresponding homologous fragmentsof CD3 epsilon of various primates, including New-World Monkeys(Marmoset, Callithrix jacchus; Saguinus oedipus; Saimiri sciureus) andOld-World Monkeys (Macaca fascicularis, also known as Cynomolgus Monkey;or Macaca mulatta, also known as Rhesus Monkey). Thus, multi-primatespecificity of the bispecific single chain antibody of the invention wasdetected. The following sequence analyses confirmed that human andprimates share a highly homologous sequence stretch at the N-terminus ofthe extracellular domain of CD3 epsilon.

The amino acid sequence of the aformentioned N-terminal fragments of CD3epsilon are depicted in SEQ ID No. 2 (human), SEQ ID No. 4 (Callithrixjacchus); SEQ ID No. 6 (Saguinus oedipus); SEQ ID No. 8 (Saimirisciureus); SEQ ID No. 427 QDGNEEMGSITQTPYQVSISGTTILTC or SEQ ID No. 428QDGNEEMGSITQTPYQVSISGTTVILT (Macaca fascicularis, also known asCynomolgus Monkey), and SEQ ID No. 429 QDGNEEMGSITQTPYHVSISGTTVILT(Macaca mulatta, also known as Rhesus Monkey).

The second binding domain of the single domain bispecific single chainantibody of the invention binds to a human or a non-chimpanzee primatetumor target antigen; more preferred it binds to the human tumor targetantigen and a non-chimpanzee primate tumor target antigen and thereforeis cross-species specific; even more preferred to the human tumor targetantigen and the macaque tumor target antigen (and therefore iscross-species specific as well). Particularly preferred, the macaquetarget antigen is the Cynomolgus monkey tumor target antigen and/or theRhesus monkey tumor target antigen. However, it is not excluded from thescope of the present invention, that the second binding domain may alsobind to tumor target antigen homologs of other species, such as to thetumor target antigen homolog in rodents. Preferably, the tumor targetantigen bound by the second binding domain of the bispecific antibody ofthe invention is EGFR, CD44v6 or CD30.

EGFR (also known as c-erb1 or HER1) belongs to the erbB receptortyrosine kinase family. When activated by binding of a ligand from theEGF family of growth factors, EGFR homodimerizes or heterodimerizes witha second EGFR or another member of the erbB receptor family,respectively, initiating a signaling cascade through mitogen-activatedprotein kinases and other transcription factors leading toproliferation, differentiation and repair (Olayioye, EMBO J. 19 (2000),3159-67). EGFR is overexpressed in many epithelial cancers, includingcolorectal, breast, lung, and head and neck cancers (Mendelsohn, J.Clin. Oncol. 21 (2003), 2787-99; Mendelsohn, J. Clin. Oncol. 20 (18,Suppl.) (2002), 1S-13S; Prewett, Clin. Cancer Res. 8 (2002), 994-1003).Overexpression and/or mutation of EGFR in malignant cells leads toconstitutive activation of kinase activity resulting in proliferation,angiogenesis, invasion, metastasis, and inhibition of apoptosis(Mendelsohn (2003, loc. cit.; Ciardiello, Clin. Cancer Res. 7 (2001),2958-70; Perez-Soler, Oncologist 9 (2004), 58-67). Monoclonal antibodiesthat target the extracellular ligand binding domain or the intracellulartyrosine kinase signaling cascade of EGFR have been shown efficacy asantitumor target (Laskin, Cancer Treat. Review 30 (2004), 1-17). Forexample, cetuximab (Erbitux) a humanized monoclonal antibody to EGFR,which competitively inhibits the extracellular domain of EGFR to inhibitligand activation of the receptor, was approved by the Food and DrugAdministration (FDA) in 2004 for the treatment of metastatic coloncancer in combination with the topoisomerase inhibitor irinotecan.

CD30 (also known as Tumor necrosis factor receptor superfamily member 8(TNFRSF8) or Lymphoid activation antigen) is expressed by activated, butnot by resting, B or T cells. In the absence of CD30 signaling,activated lymphoid cells which are not eliminated via CD95-stimulation(apoptosis) gain the ability to proliferate extensively upon secondaryencounter with antigen on parenchymal tissues, such as the pancreaticislets. Thus, CD30 signaling is understood to limit the proliferativepotential of autoreactive CD8 effector T cells, and protects the bodyagainst autoimmunity. The cDNA of CD30 was cloned by Durkop et al. (Cell68 (3), 421-427 (1992) GenBank Accession No: NM_(—)001243). CD30interacts with TRAF2 and TRAF5 and mediate the activation of nuclearfactor kappa-B. Several chronic inflammatory skin diseases areassociated with increased numbers of mast cells and increased expressionof CD30. Moreover, the lymphoid activation antigen CD30 is expressed onthe H-RS cells of classical Hodgkin's lyphoma. Currently, clinicalinvestigations are focusing on two anti-CD30 monoclonal antibodies, thehumanized SGN-30 monoclonal antibody and the fully human 5F11 monoclonalantibody. SGN-30, a chimeric anti-CD30 monoclonal antibody, hasdemonstrated antitumor activity in preclinical models of Hodgkin'slymphoma and anaplastic large cell lymphoma. MDX-060 is a fully humanIgG1k monoclonal antibody that recognizes CD30 and mediates killing ofHodgkin's lymphoma and anaplastic large cell lymphoma cell lines invitro and in xenograft tumor models (Klimm et al. Hematologica (2005) 90(12):1680).

CD44 is also known as IN, LHR, MC56, MDU2, MDU3, MIC4, Pgp1, CDW44,CSPG8, HCELL, MUTCH-I, ECMR-III or MGC10468. Accession numbers can befound in GenBank under e.g. NM_(—)000610. The human CD44 gene encodestype 1 transmembrane glycoproteins involved in cell-cell and cell-matrixinteractions. The structural heterogeneity of the gene products iscaused primarily by alternative splicing of at least 10 out of 20 exons.Certain CD44 variant isoforms, in particular those containing CD44variant domain 6 (CD44v6), have been implicated in tumourigenesis,tumour cell invasion and metastasis. CD44v6 expression in humanmalignancies (primary epithelial and nonepithelial tumours as well asmetastases) and normal tissues, and several examples of the clinical useof CD44v6-specific antibodies are reviewed in Heider et al. (CancerImmunology, Immunotherapy (2004) 53(7):567-79). In nonmalignant tissues,CD44v6 expression is essentially restricted to a subset of epithelia.Intense and homogeneous expression of CD44v6 was reported for themajority of squamous cell carcinomas and a proportion of adenocarcinomasof differing origin, but was rarely seen in nonepithelial tumours. Thisexpression pattern has made CD44v6 an attractive target forantibody-guided therapy of various types of epithelium-derived cancers.

Advantageously, the present invention provides also single domainbispecific single chain antibodies comprising a second binding domainwhich binds both to the human tumor target antigen and to the macaquetumor target antigen homolog, i.e. the homolog of a non-chimpanzeeprimate. In a preferred embodiment, the bispecific single chain antibodythus comprises a second binding domain exhibiting cross-speciesspecificity to the human and a non-chimpanzee primate tumor targetantigen. In this case, the identical bispecific single chain antibodymolecule can be used both for preclinical evaluation of safety, activityand/or pharmacokinetic profile of these binding domains in primates andas drugs in humans. Put in other words, the same molecule can be used inpreclinical animal studies as well as in clinical studies in humans.This leads to highly comparable results and a much-increased predictivepower of the animal studies compared to species-specific surrogatemolecules. Since both the CD3 and the second binding domain of thesingle domain bispecific single chain antibody of the invention arecross-species specific, i.e. reactive with the human and non-chimpanzeeprimates, it can be used both for preclinical evaluation of safety,activity and/or pharmacokinetic profile of these binding domains inprimates and—in the identical form—as drugs in humans. It will beunderstood that in a preferred embodiment, the cross-species specificityof the first and second binding domain of the antibodies of theinvention is identical.

It has been found in the present invention that it is possible togenerate a single domain bispecific single chain antibody wherein theidentical molecule can be used in preclinical animal testing, as well asclinical studies and even in therapy in human. This is due to theunexpected identification of the single domain bispecific single chainantibody, which, in addition to binding to human CD3 epsilon and tumortarget antigen, respectively, (and due to genetic similarity likely tothe chimpanzee counterpart), also binds to the homologs of said antigensof non-chimpanzee primates, including New-World Monkeys and Old-WorldMonkeys. As shown in the following examples, said single domainbispecific single chain antibody of the invention can be used astherapeutic agent or drug against various diseases, including, but notlimited, to cancer. The single domain bispecific single chain antibodyis particularly advantageous for the therapy of cancer. In view of theabove, the need to construct a surrogate single domain bispecific singlechain antibody for testing in a phylogenetic distant (from humans)species disappears. As a result, the identical molecule can be used inanimal preclinical testing as is intended to be administered to humansin clinical testing as well as following market approval and therapeuticdrug administration. The ability to use the same molecule forpreclinical animal testing as in later administration to humansvirtually eliminates, or at least greatly reduces, the danger that thedata obtained in preclinical animal testing have limited applicabilityto the human case. In short, obtaining preclinical safety data inanimals using the same molecule as will actually be administered tohumans does much to ensure the applicability of the data to ahuman-relevant scenario. In contrast, in conventional approaches usingsurrogate molecules, said surrogate molecules have to be molecularlyadapted to the animal test system used for preclinical safetyassessment. Thus, the molecule to be used in human therapy in factdiffers in sequence and also likely in structure from the surrogatemolecule used in preclinical testing in pharmacokinetic parametersand/or biological activity, with the consequence that data obtained inpreclinical animal testing have limited applicability/transferability tothe human case. The use of surrogate molecules requires theconstruction, production, purification and characterization of acompletely new construct. This leads to additional development costs andtime necessary to obtain that molecule. In sum, surrogates have to bedeveloped separately in addition to the actual drug to be used in humantherapy, so that two lines of development for two molecules have to becarried out. Therefore, a major advantage of the single domainbispecific single chain antibody of the invention exhibitingcross-species specificity described herein is that the identicalmolecule can be used for therapeutics in humans and in preclinicalanimal testing.

It is preferred that at least one of said first or second bindingdomains of the bispecific single chain antibody of the invention isCDR-grafted, humanized or human, as set forth in more detail below.Preferably, both the first and second binding domains of the bispecificsingle chain antibody of the invention are CDR-grafted, humanized orhuman. For the single domain bispecific single chain antibody of theinvention, the generation of an immune reaction against said bindingmolecules is excluded to the maximum possible extent upon administrationof the molecule to human patients.

Another major advantage of the single domain bispecific single chainantibody of the invention is its applicability for preclinical testingin various primates. The behavior of a drug candidate in animals shouldideally be indicative of the expected behavior of this drug candidateupon administration to humans. As a result, the data obtained from suchpreclinical testing should therefore generally have a highly predictivepower for the human case. However, as learned from the tragic outcome ofthe recent Phase I clinical trial on TGN1412 (a CD28 monoclonalantibody), a drug candidate may act differently in a primate speciesthan in humans: Whereas in preclinical testing of said antibody no oronly limited adverse effects have been observed in animal studiesperformed with cynomolgus monkeys, six human patients developed multipleorgan failure upon administration of said antibody (Lancet 368 (2006),2206-7). The results of these dramatic, non-desired negative eventssuggest that it may not be sufficient to limit preclinical testing toonly one (non-chimpanzee primate) species. The fact that the singledomain bispecific single chain antibody of the invention binds to aseries of New-World and Old-World Monkeys may help to overcome theproblems faced in the case mentioned above. Accordingly, the presentinvention provides means and methods for minimizing species differencesin effects when drugs for human therapy are being developed and tested.

With the cross-species specific single domain bispecific single chainantibody of the invention it is also no longer necessary to adapt thetest animal to the drug candidate intended for administration to humans,such as e.g. the creation of transgenic animals. The single domainbispecific single chain antibody of the invention exhibitingcross-species specificity according to the uses and the methods ofinvention can be directly used for preclinical testing in non-chimpanzeeprimates, without any genetic manipulation of the animals. As well knownto those skilled in the art, approaches in which the test animal isadapted to the drug candidate always bear the risk that the resultsobtained in the preclinical safety testing are less representative andpredictive for humans due to the modification of the animal. Forexample, in transgenic animals, the proteins encoded by the transgenesare often highly over-expressed. Thus, data obtained for the biologicalactivity of an antibody against this protein antigen may be limited intheir predictive value for humans in which the protein is expressed atmuch lower, more physiological levels.

A further advantage of the uses of the single domain bispecific singlechain antibody of the invention exhibiting cross-species specificity isthe fact that chimpanzees as an endangered species are avoided foranimal testing. Chimpanzees are the closest relatives to humans and wererecently grouped into the family of hominids based on the genomesequencing data (Wildman et al., PNAS 100 (2003), 7181). Therefore, dataobtained with chimpanzee is generally considered to be highly predictivefor humans. However, due to their status as endangered species, thenumber of chimpanzees, which can be used for medical experiments, ishighly restricted. As stated above, maintenance of chimpanzees foranimal testing is therefore both costly and ethically problematic. Theuses of the single domain bispecific single chain antibody of theinvention avoid both ethical objections and financial burden duringpreclinical testing without prejudicing the quality, i.e. applicability,of the animal testing data obtained. In light of this, the uses of thesingle domain bispecific single chain antibody of the invention providefor a reasonable alternative for studies in chimpanzees.

A further advantage of the single domain bispecific single chainantibody of the invention is the ability of extracting multiple bloodsamples when using it as part of animal preclinical testing, for examplein the course of pharmacokinetic animal studies. Multiple bloodextractions can be much more readily obtained with a non-chimpanzeeprimate than with lower animals, e.g. a mouse. The extraction ofmultiple blood samples allows continuous testing of blood parameters forthe determination of the biological effects induced by the single domainbispecific single chain antibody of the invention. Furthermore, theextraction of multiple blood samples enables the researcher to evaluatethe pharmacokinetic profile of the single domain bispecific single chainantibody of the invention as defined herein. In addition, potential sideeffects, which may be induced by said single domain bispecific singlechain antibody of the invention reflected in blood parameters can bemeasured in different blood samples extracted during the course of theadministration of said antibody. This allows the determination of thepotential toxicity profile of the single domain bispecific single chainantibody of the invention as defined herein.

The advantages of the single domain bispecific single chain antibody ofthe invention as defined herein exhibiting cross-species specificity maybe briefly summarized as follows:

First, the single domain bispecific single chain antibody of theinvention as defined herein used in preclinical testing is the same asthe one used in human therapy. Thus, it is no longer necessary todevelop two independent molecules, which may differ in theirpharmacokinetic properties and biological activity. This is highlyadvantageous in that e.g. the pharmacokinetic results are more directlytransferable and applicable to the human setting than e.g. inconventional surrogate approaches.

Second, the uses of the single domain bispecific single chain antibodyof the invention as defined herein for the preparation of therapeuticsin human is less cost- and labor-intensive than surrogate approaches.

Third, the single domain bispecific single chain antibody of theinvention as defined herein can be used for preclinical testing not onlyin one primate species, but in a series of different primate species,thereby limiting the risk of potential species differences betweenprimates and human.

Fourth, chimpanzee as an endangered species for animal testing can beavoided if desired.

Fifth, multiple blood samples can be extracted for extensivepharmacokinetic studies. Sixth, due to the human origin of the bindingmolecules according to a preferred embodiment of the invention thegeneration of an immune reaction against said binding molecules isminimalized when administered to human patients. Induction of an immuneresponse with antibodies specific for a drug candidate derived from anon-human species as e.g. a mouse leading to the development ofhuman-anti-mouse antibodies (HAMAs) against therapeutic molecules ofmurine origin is excluded.

Last but not least, the therapeutic use of the single domain bispecificsingle chain antibody of the invention provides a novel and inventivetherapeutic approach for cancer. The following examples clearlydemonstrate the potent recruitment of cytotoxic activity of human andmacaque effector cells against cells positive for a tumor targetantigen.

As noted herein above, the present invention provides polypeptides, i.e.bispecific single chain antibodies, comprising a first binding domainconsisting of one or two antibody variable domains capable of binding toan epitope of human and non-chimpanzee primate CD3ε chain and a secondbinding domain consisting of one or two antibody variable domainscapable of binding to a tumor target antigen, wherein the second bindingdomain preferably also binds to a tumor target antigen of a human and anon-chimpanzee primate. The advantage of bispecific single chainantibody molecules as drug candidates fulfilling the requirements of thepreferred bispecific single chain antibody of the invention is the useof such molecules in preclinical animal testing as well as in clinicalstudies and even for therapy in human. In a preferred embodiment of thecross-species specific bispecific single chain antibodies of theinvention the second binding domain binding to a cell surface antigen ishuman. In a cross-species specific bispecific molecule according to theinvention the binding domain binding to an epitope of human andnon-chimpanzee primate CD3 epsilon chain is located in the order VH-VLor VL-VH or VHH or VH at the N-terminus or the C-terminus of thebispecific molecule. Examples for cross-species specific bispecificmolecules according to the invention in different arrangements of theVH- and the VL-chain and the VHH chain consisting of one or two antibodyvariable domains in the first and the second binding domain aredescribed in the appended examples.

As used herein, a “bispecific single chain antibody” denotes a singlepolypeptide chain comprising two binding domains. For example, eachbinding domain comprises one variable region from an antibody heavychain (“VH region”), wherein the VH region of the first binding domainspecifically binds to the CD3ε molecule, and the VH region of the secondbinding domain specifically binds to a tumor target antigen.Alternatively, each binding domain comprises one VHH region, wherein theVHH region of the first binding domain specifically binds to the CD3εmolecule, and the VHH region of the second binding domain specificallybinds to a tumor target antigen. The two binding domains are optionallylinked to one another by a short polypeptide spacer. A non-limitingexample for a polypeptide spacer is Gly-Gly-Gly-Gly-Ser (G-G-G-G-S) andrepeats thereof. Each binding domain may in addition to a VH region asdescribed above comprise one variable region from an antibody lightchain (“VL region”), the VH region and VL region within each of thefirst or second binding domains being linked to one another via apolypeptide linker, for example of the type disclosed and claimed in EP623679 B1, but in any case long enough to allow the VH region and VLregion of the first binding domain and the VH region and VL region ofthe second binding domain to pair with one another such that, together,they are able to specifically bind to the respective first and secondbinding domains. Preferred formats for the bispecific single chainantibody of the invention are for example VH tumor target antigen-VLtumor target antigen-VHH CD3 or VL tumor target antigen-VH tumor targetantigen-VHH CD3 or VHH tumor target antigen-VH CD3-VL-CD3.

The term “protein” is well known in the art and describes biologicalcompounds. Proteins comprise one or more amino acid chains(polypeptides), whereby the amino acids are bound among one another viaa peptide bond. The term “polypeptide” as used herein describes a groupof molecules, which consists of more than 30 amino acids. In accordancewith the invention, the group of polypeptides comprises “proteins” aslong as the proteins consist of a single polypeptide chain. Also in linewith the definition the term “polypeptide” describes fragments ofproteins as long as these fragments consist of more than 30 amino acids.Polypeptides may further form multimers such as dimers, trimers andhigher oligomers, i.e. consisting of more than one polypeptide molecule.Polypeptide molecules forming such dimers, trimers etc. may be identicalor non-identical. The corresponding higher order structures of suchmultimers are, consequently, termed homo- or heterodimers, homo- orheterotrimers etc. An example for a hereteromultimer is an antibodymolecule, which, in its naturally occurring form, consists of twoidentical light polypeptide chains and two identical heavy polypeptidechains. The terms “polypeptide” and “protein” also refer to naturallymodified polypeptides/proteins wherein the modification is effected e.g.by post-translational modifications like glycosylation, acetylation,phosphorylation and the like. Such modifications are well known in theart.

The term “binding domain” characterizes in connection with the presentinvention a domain of a polypeptide which specifically bindsto/interacts with a given target structure/antigen/epitope. Thus, thebinding domain is an “antigen-interaction-site”. The term“antigen-interaction-site” defines, in accordance with the presentinvention, a motif of a polypeptide, which is able to specificallyinteract with a specific antigen or tumor target antigen or a specificgroup of antigens, e.g. the identical antigen or tumor target antigen indifferent species. Said binding/interaction is also understood to definea “specific recognition”. The term “specifically recognizing” means inaccordance with this invention that the antibody molecule is capable ofspecifically interacting with and/or binding to at least two, preferablyat least three, more preferably at least four amino acids of an antigen,e.g. the human CD3 antigen as defined herein. Such binding may beexemplified by the specificity of a “lock-and-key-principle”. Thus,specific motifs in the amino acid sequence of the binding domain and theantigen bind to each other as a result of their primary, secondary ortertiary structure as well as the result of secondary modifications ofsaid structure. The specific interaction of the antigen-interaction-sitewith its specific antigen may result as well in a simple binding of saidsite to the antigen. Moreover, the specific interaction of the bindingdomain/antigen-interaction-site with its specific antigen mayalternatively result in the initiation of a signal, e.g. due to theinduction of a change of the conformation of the antigen, anoligomerization of the antigen, etc. A preferred example of a bindingdomain in line with the present invention is an antibody. The bindingdomain may be a monoclonal or polyclonal antibody or derived from amonoclonal or polyclonal antibody.

The term “antibody” comprises derivatives or functional fragmentsthereof which still retain the binding specificity. Techniques for theproduction of antibodies are well known in the art and described, e.g.in Harlow and Lane “Antibodies, A Laboratory Manual”, Cold Spring HarborLaboratory Press, 1988 and Harlow and Lane “Using Antibodies: ALaboratory Manual” Cold Spring Harbor Laboratory Press, 1999. The term“antibody” also comprises immunoglobulins (Ig's) of different classes(i.e. IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2etc.).

The definition of the term “antibody” also includes embodiments such aschimeric, single chain and humanized antibodies, as well as antibodyfragments, like, inter alia, Fab fragments. Antibody fragments orderivatives further comprise F(ab′)₂, Fv, scFv fragments or singledomain antibodies, single variable domain antibodies or immunoglobulinsingle variable domain comprising merely one variable domain, whichmight be VH or VL, that specifically bind to an antigen or epitopeindependently of other V regions or domains; see, for example, Harlowand Lane (1988) and (1999), loc. cit. Such immunoglobulin singlevariable domain encompasses not only an isolated antibody singlevariable domain polypeptide, but also larger polypeptides that compriseone or more monomers of an antibody single variable domain polypeptidesequence.

Various procedures are known in the art and may be used for theproduction of such antibodies and/or fragments. Thus, the (antibody)derivatives can also be produced by peptidomimetics. Further, techniquesdescribed for the production of single chain antibodies (see, interalia, U.S. Pat. No. 4,946,778) can be adapted to produce single chainantibodies specific for elected polypeptide(s). Also, transgenic animalsmay be used to express humanized antibodies specific for polypeptidesand fusion proteins of this invention. For the preparation of monoclonalantibodies, any technique, providing antibodies produced by continuouscell line cultures can be used. Examples for such techniques include thehybridoma technique (Köhler and Milstein Nature 256 (1975), 495-497),the trioma technique, the human B-cell hybridoma technique (Kozbor,Immunology Today 4 (1983), 72) and the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96). Surface plasmonresonance as employed in the BIAcore system can be used to increase theefficiency of phage antibodies which bind to an epitope of a targetpolypeptide, such as CD3 epsilon or a tumor target antigen (Schier,Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol.Methods 183 (1995), 7-13). It is also envisaged in the context of thisinvention that the term “antibody” comprises antibody constructs, whichmay be expressed in a host as described herein below, e.g. antibodyconstructs which may be transfected and/or transduced via, inter alia,viruses or plasmid vectors.

The term “specific interaction” as used in accordance with the presentinvention means that the binding domain does not or does notsignificantly cross-react with polypeptides which have similar structureas those bound by the binding domain, and which might be expressed bythe same cells as the polypeptide of interest. Cross-reactivity of apanel of binding domains under investigation may be tested, for example,by assessing binding of said panel of binding domains under conventionalconditions (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory Press, 1988 and Using Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1999). Examplesfor the specific interaction of a binding domain with a specific antigencomprise the specificity of a ligand for its receptor. Said definitionparticularly comprises the interaction of ligands, which induce a signalupon binding to its specific receptor. Examples for said interaction,which is also particularly comprised by said definition, is theinteraction of an antigenic determinant (epitope) with the bindingdomain (antigenic binding site) of an antibody.

The term “cross-species specificity” or “interspecies specificity” asused herein means binding of a binding domain described herein to thesame target molecule in humans and non-chimpanzee primates. Thus,“cross-species specificity” or “interspecies specificity” is to beunderstood as an interspecies reactivity to the same molecule “X” (i.e.the homolog) expressed in different species, but not to a molecule otherthan “X”. Cross-species specificity of a monoclonal antibody recognizinge.g. human CD3 epsilon, to a non-chimpanzee primate CD3 epsilon, e.g.macaque CD3 epsilon, can be determined, for instance, by FACS analysis.The FACS analysis is carried out in a way that the respective monoclonalantibody is tested for binding to human and non-chimpanzee primatecells, e.g. macaque cells, expressing said human and non-chimpanzeeprimate CD3 epsilon antigens, respectively. An appropriate assay isshown in the following examples. The above-mentioned subject matterapplies mutatis mutandis for the tumor target antigen: Cross-speciesspecificity of a monoclonal antibody recognizing human tumor targetantigen, to a non-chimpanzee primate tumor target antigen, e.g. macaquetumor target antigen, can be determined, for instance, by FACS analysis.The FACS analysis is carried out in a way that the respective monoclonalantibody is tested for binding to human and non-chimpanzee primatecells, e.g. macaque cells, expressing said human and non-chimpanzeeprimate tumor target antigens, respectively. Preferably, the tumortarget antigen bound by the second binding domain of the bispecificantibody of the invention is EGFR, CD44v6 or CD30.

As used herein, CD3 epsilon denotes a molecule expressed as part of theT cell receptor and has the meaning as typically ascribed to it in theprior art. In human, it encompasses in individual or independentlycombined form all known CD3 subunits, for example CD3 epsilon, CD3delta, CD3 gamma, CD3 zeta, CD3 alpha and CD3 beta. The non-chimpanzeeprimate, non-human CD3 antigens as referred to herein are, for example,Macaca fascicularis CD3 and Macaca mulatta CD3. In Macaca fascicularis,it encompasses CD3 epsilon FN-18 negative and CD3 epsilon FN-18positive, CD3 gamma and CD3 delta. In Macaca mulatta, it encompasses CD3epsilon, CD3 gamma and CD3 delta. Preferably, said CD3 as used herein isCD3 epsilon.

The human CD3 epsilon is indicated in GenBank Accession No. NM_(—)000733and comprises SEQ ID NO. 1. The human CD3 gamma is indicated in GenBankAccession NO. NM_(—)000073. The human CD3 delta is indicated in GenBankAccession No. NM_(—)000732.

The CD3 epsilon “FN-18 negative” of Macaca fascicularis (i.e. CD3epsilon not recognized by monoclonal antibody FN-18 due to apolymorphism as set forth above) is indicated in GenBank Accession No.AB073994.

The CD3 epsilon “FN-18 positive” of Macaca fascicularis (i.e. CD3epsilon recognized by monoclonal antibody FN-18) is indicated in GenBankAccession No. AB073993. The CD3 gamma of Macaca fascicularis isindicated in GenBank Accession No. AB073992. The CD3 delta of Macacafascicularis is indicated in GenBank Accession No. AB073991.

The nucleic acid sequences and amino acid sequences of the respectiveCD3 epsilon, gamma and delta homologs of Macaca mulatta can beidentified and isolated by recombinant techniques described in the art(Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold SpringHarbor Laboratory Press, 3^(rd) edition 2001). This applies mutatismutandis to the CD3 epsilon, gamma and delta homologs of othernon-chimpanzee primates as defined herein. The identification of theamino acid sequence of Callithrix jacchus, Saimiri sciureus and Saguinusoedipus is described in the appended examples. The amino acid sequenceof the extracellular domain of the CD3 epsilon of Callithrix jacchus isdepicted in SEQ ID NO: 3, the one of Saguinus oedipus is depicted in SEQID NO: 5 and the one of Saimiri sciureus is depicted in SEQ ID NO: 7.

The accession number of EGFR, CD44v6 and CD30 have been indicated above.On the basis of this sequence information it is possible for the personskilled in the art without any inventive ado to clone (and express) themacaque tumor target antigen. For example, the human EGFR cDNA or afragment thereof indicated in the above-mentioned GenBank Accession No.can be used as a hybridization probe in order to screen a macaque cDNAlibrary (e.g. a cDNA library of Cynomolgus monkey or Rhesus monkey)under appropriate hybridization conditions. Recombinant techniques andscreening methods (including hybridization approaches) in molecularbiology are described e.g. in Sambrook et al. Molecular Cloning: ALaboratory Manual; Cold Spring Harbor Laboratory Press, 3^(rd) edition2001.

In line with the above, the term “epitope” defines an antigenicdeterminant, which is specifically bound/identified by a binding domainas defined herein. The binding domain may specifically bind to/interactwith conformational or continuous epitopes, which are unique for thetarget structure, e.g. the human and non-chimpanzee primate CD3 epsilonchain or the human and non-chimpanzee primate tumor target antigen. Aconformational or discontinuous epitope is characterized for polypeptideantigens by the presence of two or more discrete amino acid residueswhich are separated in the primary sequence, but come together on thesurface of the molecule when the polypeptide folds into the nativeprotein/antigen (Sela, (1969) Science 166, 1365 and Laver, (1990) Cell61, 553-6). The two or more discrete amino acid residues contributing tothe epitope are present on separate sections of one or more polypeptidechain(s). These residues come together on the surface of the moleculewhen the polypeptide chain(s) fold(s) into a three-dimensional structureto constitute the epitope. In contrast, a continuous or linear epitopeconsists of two or more discrete amino acid residues, which are presentin a single linear segment of a polypeptide chain. Within the presentinvention, a “context-dependent” CD3 epitope refers to the conformationof said epitope. Such a context-dependent epitope, localized on theepsilon chain of CD3, can only develop its correct conformation if it isembedded within the rest of the epsilon chain and held in the rightposition by heterodimerization of the epsilon chain with either CD3gamma or delta chain. In contrast, a context-independent CD3 epitope asprovided herein refers to an N-terminal 1-27 amino acid residuepolypeptide or a functional fragment thereof of CD3 epsilon. ThisN-terminal 1-27 amino acid residue polypeptide or a functional fragmentthereof maintains its three-dimensional structural integrity and correctconformation when taken out of its native environment in the CD3complex. The context-independency of the N-terminal 1-27 amino acidresidue polypeptide or a functional fragment thereof, which is part ofthe extracellular domain of CD3 epsilon, represents, thus, an epitopewhich is completely different to the epitopes of CD3 epsilon describedin connection with a method for the preparation of human bindingmolecules in WO 2004/106380. Said method used solely expressedrecombinant CD3 epsilon. The conformation of this solely expressedrecombinant CD3 epsilon differed from that adopted in its natural form,that is, the form in which the CD3 epsilon subunit of the TCR/CD3complex exists as part of a noncovalent complex with either the CD3delta or the CD3-gamma subunit of the TCR/CD3 complex. When such solelyexpressed recombinant CD3 epsilon protein is used as an antigen forselection of antibodies from an antibody library, antibodies specificfor this antigen are identified from the library although such a librarydoes not contain antibodies with specificity forself-antigens/autoantigens. This is due to the fact that solelyexpressed recombinant CD3 epsilon protein does not exist in vivo; it isnot an autoantigen. Consequently, subpopulations of B cells expressingantibodies specific for this protein have not been depleted in vivo; anantibody library constructed from such B cells would contain geneticmaterial for antibodies specific for solely expressed recombinant CD3epsilon protein.

However, since the context-independent N-terminal 1-27 amino acidresidue polypeptide or a functional fragment thereof is an epitope,which folds in its native form, binding domains in line with the presentinvention cannot be identified by methods based on the approachdescribed in WO 2004/106380. Therefore, it could be verified in teststhat binding molecules as disclosed in WO 2004/106380 are not capable ofbinding to the N-terminal 1-27 amino acid residues of the CD3 epsilonchain. Hence, conventional anti-CD3 binding molecules or anti-CD3antibody molecules (e.g. as disclosed in WO 99/54440) bind CD3 epsilonchain at a position which is more C-terminally located than thecontext-independent N-terminal 1-27 amino acid residue polypeptide or afunctional fragment provided herein. Prior art antibody molecules OKT3and UCHT-1 have also a specificity for the epsilon-subunit of theTCR/CD3 complex between amino acid residues 35 to 85 and, accordingly,the epitope of these antibodies is also more C-terminally located. Inaddition, UCHT-1 binds to the CD3 epsilon chain in a region betweenamino acid residues 43 to 77 (Tunnacliffe, Int. Immunol. 1 (1989),546-50; Kjer-Nielsen, PNAS 101, (2004), 7675-7680; Salmeron, J. Immunol.147 (1991), 3047-52). Therefore, prior art anti-CD3 molecules do notbind to and are not directed against the herein definedcontext-independent N-terminal 1-27 amino acid residue epitope (or afunctional fragment thereof). In particular, the state of the art failsto provide anti-CD3 molecules which specifically binds to thecontext-independent N-terminal 1-27 amino acid residue epitope and whichare cross-species specific, i.e. bind to human and non-chimpanzeeprimate CD3 epsilon.

For the generation of a binding domain consisting of one or two antibodyvariable domains comprised in a bispecific single chain antibodymolecule of the invention, e.g. monoclonal antibodies binding to boththe human and non-chimpanzee primate CD3 epsilon (e.g. macaque CD3epsilon) or monoclonal antibodies binding to the human and/ornon-chimpanzee primate tumor target antigen can be used.

As used herein, “human” and “man” refers to the species Homo sapiens. Asfar as the medical uses of the constructs described herein areconcerned, human patients are to be treated with the same molecule.

It is preferred that at least one of said first or second bindingdomains of the bispecific single chain antibody of the invention isCDR-grafted, humanized or human. Preferably, both the first and secondbinding domains of the bispecific single chain antibody of the inventionare CDR-grafted, humanized or human.

The term “human” antibody as used herein is to be understood as meaningthat the bispecific single chain antibody as defined herein, comprises(an) amino acid sequence(s) contained in the human germline antibodyrepertoire. For the purposes of definition herein, said bispecificsingle chain antibody may therefore be considered human if it consistsof such (a) human germline amino acid sequence(s), i.e. if the aminoacid sequence(s) of the bispecific single chain antibody in question is(are) identical to (an) expressed human germline amino acid sequence(s).A bispecific single chain antibody as defined herein may also beregarded as human if it consists of (a) sequence(s) that deviate(s) fromits (their) closest human germline sequence(s) by no more than would beexpected due to the imprint of somatic hypermutation. Additionally, theantibodies of many non-human mammals, for example rodents such as miceand rats, comprise VH CDR3 amino acid sequences which one may expect toexist in the expressed human antibody repertoire as well. Any suchsequence(s) of human or non-human origin which may be expected to existin the expressed human repertoire would also be considered “human” forthe purposes of the present invention.

As used herein, the term “humanized”, “humanization”, “human-like” orgrammatically related variants thereof are used interchangeably to referto a bispecific single chain antibody comprising in at least one of itsbinding domains at least one complementarity determining region (“CDR”)from a non-human antibody or fragment thereof. Humanization approachesare described for example in WO 91/09968 and U.S. Pat. No. 6,407,213. Asnon-limiting examples, the term encompasses the case in which a variableregion of at least one binding domain comprises a single CDR region, forexample the third CDR region of the VH(CDRH3), from another non-humananimal, for example a rodent, as well as the case in which a or bothvariable region/s comprise at each of their respective first, second andthird CDRs the CDRs from said non-human animal. In the event that allCDRs of a binding domain of the bispecific single chain antibody havebeen replaced by their corresponding equivalents from, for example, arodent, one typically speaks of “CDR-grafting”, and this term is to beunderstood as being encompassed by the term “humanized” or grammaticallyrelated variants thereof as used herein. The term “humanized” orgrammatically related variants thereof also encompasses cases in which,in addition to replacement of one or more CDR regions within a VH and/orVL of the first and/or second binding domain further mutation/s (e.g.substitutions) of at least one single amino acid residue/s within theframework (“FR”) regions between the CDRs has/have been effected suchthat the amino acids at that/those positions correspond/s to the aminoacid/s at that/those position/s in the animal from which the CDR regionsused for replacement is/are derived. As is known in the art, suchindividual mutations are often made in the framework regions followingCDR-grafting in order to restore the original binding affinity of thenon-human antibody used as a CDR-donor for its target molecule. The term“humanized” may further encompass (an) amino acid substitution(s) in theCDR regions from a non-human animal to the amino acid(s) of acorresponding CDR region from a human antibody, in addition to the aminoacid substitutions in the framework regions as described above.

As used herein, the term “homolog” or “homology” is to be understood asfollows: Homology among proteins and DNA is often concluded on the basisof sequence similarity, especially in bioinformatics. For example, ingeneral, if two or more genes have highly similar DNA sequences, it islikely that they are homologous. But sequence similarity may arise fromdifferent ancestors: short sequences may be similar by chance, andsequences may be similar because both were selected to bind to aparticular protein, such as a transcription factor. Such sequences aresimilar but not homologous. Sequence regions that are homologous arealso called conserved. This is not to be confused with conservation inamino acid sequences in which the amino acid at a specific position haschanged but the physio-chemical properties of the amino acid remainunchanged. Homologous sequences are of two types: orthologous andparalogous. Homologous sequences are orthologous if they were separatedby a speciation event: when a species diverges into two separatespecies, the divergent copies of a single gene in the resulting speciesare said to be orthologous. Orthologs, or orthologous genes, are genesin different species that are similar to each other because theyoriginated from a common ancestor. The strongest evidence that twosimilar genes are orthologous is the result of a phylogenetic analysisof the gene lineage. Genes that are found within one clade areorthologs, descended from a common ancestor. Orthologs often, but notalways, have the same function. Orthologous sequences provide usefulinformation in taxonomic classification studies of organisms. Thepattern of genetic divergence can be used to trace the relatedness oforganisms. Two organisms that are very closely related are likely todisplay very similar DNA sequences between two orthologs. Conversely, anorganism that is further removed evolutionarily from another organism islikely to display a greater divergence in the sequence of the orthologsbeing studied. Homologous sequences are paralogous if they wereseparated by a gene duplication event: if a gene in an organism isduplicated to occupy two different positions in the same genome, thenthe two copies are paralogous. A set of sequences that are paralogousare called paralogs of each other. Paralogs typically have the same orsimilar function, but sometimes do not: due to lack of the originalselective pressure upon one copy of the duplicated gene, this copy isfree to mutate and acquire new functions. An example can be found inrodents such as rats and mice. Rodents have a pair of paralogous insulingenes, although it is unclear if any divergence in function hasoccurred. Paralogous genes often belong to the same species, but this isnot necessary: for example, the hemoglobin gene of humans and themyoglobin gene of chimpanzees are paralogs. This is a common problem inbioinformatics: when genomes of different species have been sequencedand homologous genes have been found, one can not immediately concludethat these genes have the same or similar function, as they could beparalogs whose function has diverged.

As used herein, a “non-chimpanzee primate” or “non-chimp primate” orgrammatical variants thereof refers to any primate animal (i.e. nothuman) other than chimpanzee, i.e. other than an animal of belonging tothe genus Pan, and including the species Pan paniscus and Pantroglodytes, also known as Anthropopithecus troglodytes or Simiasatyrus. It will be understood, however, that it is possible that theantibodies of the invention can also bind with their first and/or secondbinding domain to the respective epitopes/fragments etc. of saidchimpanzees. The intention is merely to avoid animal tests which arecarried out with chimpanzees, if desired. It is thus also envisaged thatin another embodiment the antibodies of the present invention also bindwith their first and/or second binding domain to the respective epitopesof chimpanzees. A “primate”, “primate species”, “primates” orgrammatical variants thereof denote/s an order of eutherian mammalsdivided into the two suborders of prosimians and anthropoids andcomprising apes, monkeys and lemurs. Specifically, “primates” as usedherein comprises the suborder Strepsirrhini (non-tarsier prosimians),including the infraorder Lemuriformes (itself including thesuperfamilies Chemogaleoidea and Lemuroidea), the infraorderChiromyiformes (itself including the family Daubentoniidae) and theinfraorder Lorisiformes (itself including the families Lorisidae andGalagidae). “Primates” as used herein also comprises the suborderHaplorrhini, including the infraorder Tarsiiformes (itself including thefamily Tarsiidae), the infraorder Simiiformes (itself including thePlatyrrhini, or New-World monkeys, and the Catarrhini, including theCercopithecidea, or Old-World Monkeys).

The non-chimpanzee primate species may be understood within the meaningof the invention to be a lemur, a tarsier, a gibbon, a marmoset(belonging to New-World Monkeys of the family Cebidae) or an Old-WorldMonkey (belonging to the superfamily Cercopithecoidea).

As used herein, an “Old-World Monkey” comprises any monkey falling inthe superfamily Cercopithecoidea, itself subdivided into the families:the Cercopithecinae, which are mainly African but include the diversegenus of macaques which are Asian and North African; and the Colobinae,which include most of the Asian genera but also the African colobusmonkeys.

Specifically, within the subfamily Cercopithecinae, an advantageousnon-chimpanzee primate may be from the Tribe Cercopithecini, within thegenus Allenopithecus (Allen's Swamp Monkey, Allenopithecusnigroviridis); within the genus Miopithecus (Angolan Talapoin,Miopithecus talapoin; Gabon Talapoin, Miopithecus ogouensis); within thegenus Erythrocebus (Patas Monkey, Erythrocebus patas); within the genusChlorocebus (Green Monkey, Chlorocebus sabaceus; Grivet, Chlorocebusaethiops; Bale Mountains Vervet, Chlorocebus djamdjamensis; TantalusMonkey, Chlorocebus tantalus; Vervet Monkey, Chlorocebus pygerythrus;Malbrouck, Chlorocebus cynosuros); or within the genus Cercopithecus(Dryas Monkey or Salongo Monkey, Cercopithecus dryas; Diana Monkey,Cercopithecus diana; Roloway Monkey, Cercopithecus roloway; GreaterSpot-nosed Monkey, Cercopithecus nictitans; Blue Monkey, Cercopithecusmitis; Silver Monkey, Cercopithecus doggetti; Golden Monkey,Cercopithecus kandti; Sykes's Monkey, Cercopithecus albogularis; MonaMonkey, Cercopithecus mona; Campbell's Mona Monkey, Cercopithecuscampbelli; Lowe's Mona Monkey, Cercopithecus lowei; Crested Mona Monkey,Cercopithecus pogonias; Wolfs Mona Monkey, Cercopithecus wolfi; Dent'sMona Monkey, Cercopithecus denti; Lesser Spot-nosed Monkey,Cercopithecus petaurista; White-throated Guenon, Cercopithecuserythrogaster; Sclater's Guenon, Cercopithecus sclateri; Red-earedGuenon, Cercopithecus erythrotis; Moustached Guenon, Cercopithecuscephus; Red-tailed Monkey, Cercopithecus ascanius; L'Hoest's Monkey,Cercopithecus lhoesti; Preuss's Monkey, Cercopithecus preussi;Sun-tailed Monkey, Cercopithecus solatus; Hamlyn's Monkey or Owl-facedMonkey, Cercopithecus hamlyni; De Brazza's Monkey, Cercopithecusneglectus).

Alternatively, an advantageous non-chimpanzee primate, also within thesubfamily Cercopithecinae but within the Tribe Papionini, may be fromwithin the genus Macaca (Barbary Macaque, Macaca sylvanus; Lion-tailedMacaque, Macaca silenus; Southern Pig-tailed Macaque or Beruk, Macacanemestrina; Northern Pig-tailed Macaque, Macaca leonina; Pagai IslandMacaque or Bokkoi, Macaca pagensis; Siberut Macaque, Macaca siberu; MoorMacaque, Macaca maura; Booted Macaque, Macaca ochreata; Tonkean Macaque,Macaca tonkeana; Heck's Macaque, Macaca hecki; Gorontalo Macaque, Macacanigriscens; Celebes Crested Macaque or Black “Ape”, Macaca nigra;Cynomolgus monkey or Crab-eating Macaque or Long-tailed Macaque or Kera,Macaca fascicularis; Stump-tailed Macaque or Bear Macaque, Macacaarctoides; Rhesus Macaque, Macaca mulatta; Formosan Rock Macaque, Macacacyclopis; Japanese Macaque, Macaca fuscata; Toque Macaque, Macacasinica; Bonnet Macaque, Macaca radiata; Barbary Macaque, Macacasylvanmus; Assam Macaque, Macaca assamensis; Tibetan Macaque orMilne-Edwards' Macaque, Macaca thibetana; Arunachal Macaque or Munzala,Macaca munzala); within the genus Lophocebus (Gray-cheeked Mangabey,Lophocebus albigena; Lophocebus albigena albigena; Lophocebus albigenaosmani; Lophocebus albigena johnstoni; Black Crested Mangabey,Lophocebus aterrimus; Opdenbosch's Mangabey, Lophocebus opdenboschi;Highland Mangabey, Lophocebus kipunji); within the genus Papio(Hamadryas Baboon, Papio hamadryas; Guinea Baboon, Papio papio; OliveBaboon, Papio anubis; Yellow Baboon, Papio cynocephalus; Chacma Baboon,Papio ursinus); within the genus Theropithecus (Gelada, Theropithecusgelada); within the genus Cercocebus (Sooty Mangabey, Cercocebus atys;Cercocebus atys atys; Cercocebus atys lunulatus; Collared Mangabey,Cercocebus torquatus; Agile Mangabey, Cercocebus agilis; Golden-belliedMangabey, Cercocebus chrysogaster; Tana River Mangabey, Cercocebusgaleritus; Sanje Mangabey, Cercocebus sanjei); or within the genusMandrillus (Mandrill, Mandrillus sphinx; Drill, Mandrillus leucophaeus).

Most preferred is Macaca fascicularis (also known as Cynomolgus monkeyand, therefore, in the Examples named “Cynomolgus”) and Macaca mulatta(rhesus monkey, named “rhesus”).

Within the subfamily Colobinae, an advantageous non-chimpanzee primatemay be from the African group, within the genus Colobus (Black Colobus,Colobus satanas; Angola Colobus, Colobus angolensis; King Colobus,Colobus polykomos; Ursine Colobus, Colobus vellerosus; Mantled Guereza,Colobus guereza); within the genus Piliocolobus (Western Red Colobus,Piliocolobus badius; Piliocolobus badius badius; Piliocolobus badiustemminckii; Piliocolobus badius waldronae; Pennant's Colobus,Piliocolobus pennantii; Piliocolobus pennantii pennantii; Piliocolobuspennantii epieni; Piliocolobus pennantii bouvieri; Preuss's Red Colobus,Piliocolobus preussi; Thollon's Red Colobus, Piliocolobus tholloni;Central African Red Colobus, Piliocolobus foai; Piliocolobus foai foai;Piliocolobus foai ellioti; Piliocolobus foai oustaleti; Piliocolobusfoai semlikiensis; Piliocolobus foai parmentierorum; Ugandan RedColobus, Piliocolobus tephrosceles; Uzyngwa Red Colobus, Piliocolobusgordonorum; Zanzibar Red Colobus, Piliocolobus kirkii; Tana River RedColobus, Piliocolobus rufomitratus); or within the genus Procolobus(Olive Colobus, Procolobus verus). Within the subfamily Colobinae, anadvantageous non-chimpanzee primate may alternatively be from the Langur(leaf monkey) group, within the genus Semnopithecus (Nepal Gray Langur,Semnopithecus schistaceus; Kashmir Gray Langur, Semnopithecus ajax;Tarai Gray Langur, Semnopithecus hector; Northern Plains Gray Langur,Semnopithecus entellus; Black-footed Gray Langur, Semnopithecushypoleucos; Southern Plains Gray Langur, Semnopithecus dussumieri;Tufted Gray Langur, Semnopithecus priam); within the T. vetulus group orthe genus Trachypithecus (Purple-faced Langur, Trachypithecus vetulus;Nilgiri Langur, Trachypithecus johnii); within the T. cristatus group ofthe genus Trachypithecus (Javan Lutung, Trachypithecus auratus; SilveryLeaf Monkey or Silvery Lutung, Trachypithecus cristatus; IndochineseLutung, Trachypithecus germaini; Tenasserim Lutung, Trachypithecusbarbei); within the T. obscurus group of the genus Trachypithecus (DuskyLeaf Monkey or Spectacled Leaf Monkey, Trachypithecus obscurus; Phayre'sLeaf Monkey, Trachypithecus phayrei); within the T. pileatus group ofthe genus Trachypithecus (Capped Langur, Trachypithecus pileatus;Shortridge's Langur, Trachypithecus shortridgei; Gee's Golden Langur,Trachypithecus geei); within the T. francoisi group of the genusTrachypithecus (Francois' Langur, Trachypithecus francoisi; HatinhLangur, Trachypithecus hatinhensis; White-headed Langur, Trachypithecuspoliocephalus; Laotian Langur, Trachypithecus laotum; Delacour's Langur,Trachypithecus delacouri; Indochinese Black Langur, Trachypithecusebenus); or within the genus Presbytis (Sumatran Surili, Presbytismelalophos; Banded Surili, Presbytis femoralis; Sarawak Surili,Presbytis chrysomelas; White-thighed Surili, Presbytis siamensis;White-fronted Surili, Presbytis frontata; Javan Surili, Presbytiscomata; Thomas's Langur, Presbytis thomasi; Hose's Langur, Presbytishosei; Maroon Leaf Monkey, Presbytis rubicunda; Mentawai Langur or Joja,Presbytis potenziani; Natuna Island Surili, Presbytis natunae).

Within the subfamily Colobinae, an advantageous non-chimpanzee primatemay alternatively be from the Odd-Nosed group, within the genusPygathrix (Red-shanked Douc, Pygathrix nemaeus; Black-shanked Douc,Pygathrix nigripes; Gray-shanked Douc, Pygathrix cinerea); within thegenus Rhinopithecus (Golden Snub-nosed Monkey, Rhinopithecus roxellana;Black Snub-nosed Monkey, Rhinopithecus bieti; Gray Snub-nosed Monkey,Rhinopithecus brelichi; Tonkin Snub-nosed Langur, Rhinopithecusavunculus); within the genus Nasalis (Proboscis Monkey, Nasalislarvatus); or within the genus Simias (Pig-tailed Langur, Simiasconcolor).

As used herein, the term “marmoset” denotes any New-World Monkeys of thegenus Callithrix, for example belonging to the Atlantic marmosets ofsubgenus Callithrix (sic!) (Common Marmoset, Callithrix (Callithrix)jacchus; Black-tufted Marmoset, Callithrix (Callithrix) penicillata;Wied's Marmoset, Callithrix (Callithrix) kuhlii; White-headed Marmoset,Callithrix (Callithrix) geoffroyi; Buffy-headed Marmoset, Callithrix(Callithrix) flaviceps; Buffy-tufted Marmoset, Callithrix (Callithrix)aurita); belonging to the Amazonian marmosets of subgenus Mico (RioAcari Marmoset, Callithrix (Mico) acariensis; Manicore Marmoset,Callithrix (Mico) manicorensis; Silvery Marmoset, Callithrix (Mico)argentata; White Marmoset, Callithrix (Mico) leucippe; Emilia'sMarmoset, Callithrix (Mico) emiliae; Black-headed Marmoset, Callithrix(Mico) nigriceps; Marca's Marmoset, Callithrix (Mico)marcai;Black-tailed Marmoset, Callithrix (Mico) melanura; Santarem Marmoset,Callithrix (Mico) humeralifera; Maues Marmoset, Callithrix (Mico)mauesi; Gold-and-white Marmoset, Callithrix (Mico) chrysoleuca;Hershkovitz's Marmoset, Callithrix (Mico) intermedia; Satéré Marmoset,Callithrix (Mico) saterei); Roosmalens' Dwarf Marmoset belonging to thesubgenus Callibella (Callithrix (Callibella) humilis); or the PygmyMarmoset belonging to the subgenus Cebuella (Callithrix (Cebuella)pygmaea).

Other genera of the New-World Monkeys comprise tamarins of the genusSaguinus (comprising the S. oedipus-group, the S. midas group, the S.nigricollis group, the S. mystax group, the S. bicolor group and the S.inustus group) and squirrel monkeys of the genus Samiri (e.g. Saimirisciureus, Saimiri oerstedii, Saimiri ustus, Saimiri boliviensis, Saimirivanzolini)

In a preferred embodiment of the bispecific single chain antibodymolecule of the invention, the non-chimpanzee primate is an old worldmonkey. In a more preferred embodiment of the polypeptide, the old worldmonkey is a monkey of the Papio genus Macaque genus. Most preferably,the monkey of the Macaque genus is Assamese macaque (Macaca assamensis),Barbary macaque (Macaca sylvanus), Bonnet macaque (Macaca radiata),Booted or Sulawesi-Booted macaque (Macaca ochreata), Sulawesi-crestedmacaque (Macaca nigra), Formosan rock macaque (Macaca cyclopsis),Japanese snow macaque or Japanese macaque (Macaca fuscata), Cynomologusmonkey or crab-eating macaque or long-tailed macaque or Java macaque(Macaca fascicularis), Lion-tailed macaque (Macaca silenus), Pigtailedmacaque (Macaca nemestrina), Rhesus macaque (Macaca mulatta), Tibetanmacaque (Macaca thibetana), Tonkean macaque (Macaca tonkeana), Toquemacaque (Macaca sinica), Stump-tailed macaque or Red-faced macaque orBear monkey (Macaca arctoides), or Moor macaque (Macaca maurus). Mostpreferably, the monkey of the Papio genus is Hamadryas Baboon, Papiohamadryas; Guinea Baboon, Papio papio; Olive Baboon, Papio anubis;Yellow Baboon, Papio cynocephalus; Chacma Baboon, Papio ursinus.

In an alternatively preferred embodiment of the bispecific single chainantibody molecule of the invention, the non-chimpanzee primate is a newworld monkey. In a more preferred embodiment of the polypeptide, the newworld monkey is a monkey of the Callithrix genus (marmoset), theSaguinus genus or the Samiri genus. Most preferably, the monkey of theCallithrix genus is Callithrix jacchus, the monkey of the Saguinus genusis Saguinus oedipus and the monkey of the Samiri genus is Saimirisciureus.

The term “cell surface antigen” as used herein denotes a molecule, whichis displayed on the surface of a cell. In most cases, this molecule willbe located in or on the plasma membrane of the cell such that at leastpart of this molecule remains accessible from outside the cell intertiary form. A non-limiting example of a cell surface molecule, whichis located in the plasma membrane is a transmembrane protein comprising,in its tertiary conformation, regions of hydrophilicity andhydrophobicity. Here, at least one hydrophobic region allows the cellsurface molecule to be embedded, or inserted in the hydrophobic plasmamembrane of the cell while the hydrophilic regions extend on either sideof the plasma membrane into the cytoplasm and extracellular space,respectively. Non-limiting examples of cell surface molecules which arelocated on the plasma membrane are proteins which have been modified ata cysteine residue to bear a palmitoyl group, proteins modified at aC-terminal cysteine residue to bear a farnesyl group or proteins whichhave been modified at the C-terminus to bear a glycosyl phosphatidylinositol (“GPI”) anchor. These groups allow covalent attachment ofproteins to the outer surface of the plasma membrane, where they remainaccessible for recognition by extracellular molecules such asantibodies. An example of cell surface antigens is CD3 epsilon.

In light of this, tumor target antigens can also be characterized as atumor antigesn. The term “tumor target antigen” as used herein may beunderstood as those antigens that are presented on tumor cells. Theseantigens can be presented on the cell surface with an extracellularpart, which is often combined with a transmembrane and cytoplasmic partof the molecule. These antigens can sometimes be presented only by tumorcells and never by the normal ones. Tumor antigens can be exclusivelyexpressed on tumor cells or might represent a tumor specific mutationcompared to normal cells. In this case, they are called tumor-specificantigens. More common are antigens that are presented by tumor cells andnormal cells, and they are called tumor-associated antigens. Thesetumor-associated antigens can be overexpressed compared to normal cellsor are accessible for antibody binding in tumor cells due to the lesscompact structure of the tumor tissue compared to normal tissue.Preferably, the tumor target antigen bound by the second binding domainof the bispecific antibody of the invention is EGFR, CD44v6 or CD30.

As described herein above the bispecific single chain antibody moleculeof the invention binds with the first binding domain to an epitope ofhuman and non-chimpanzee primate CD3ε (epsilon) chain, wherein theepitope is part of an amino acid sequence comprised in the groupconsisting of 27 amino acid residues as depicted in SEQ ID NOs. 2, 4, 6,or 8 or a functional fragment thereof.

In line with the present invention it is preferred for the bispecificsingle chain antibody molecule of the invention that said epitope ispart of an amino acid sequence comprising 26, 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids.

More preferably, wherein said epitope comprises at least the amino acidsequence Gln-Asp-Gly-Asn-Glu (Q-D-G-N-E).

Within the present invention, a functional fragment of the N-terminal1-27 amino acid residues means that said functional fragment is still acontext-independent epitope maintaining its three-dimensional structuralintegrity when taken out of its native environment in the CD3 complex(and fused to a heterologous amino acid sequence such as EpCAM or animmunoglobulin Fc part, e.g. as shown in Example 3.1). The maintenanceof the three-dimensional structure within the 27 amino acid N-terminalpolypeptide or functional fragment thereof of CD3 epsilon can be usedfor the generation of binding domains which bind to the N-terminal CD3epsilon polypeptide fragment in vitro and to the native (CD3 epsilonsubunit of the) CD3 complex on T cells in vivo with the same bindingaffinity. Within the present invention, a functional fragment of theN-terminal 1-27 amino acid residues means that CD3 binding domainsprovided herein can still bind to such functional fragments in acontext-independent manner. The person skilled in the art is aware ofmethods for epitope mapping to determine which amino acid residues of anepitope are recognized by such anti-CD3 binding domains (e.g. alaninescanning; see appended examples).

Within the present invention it is further preferred that the secondbinding domain binds to the human tumor target antigen and/or anon-chimpanzee primate tumor target antigen. Particularly preferred, thesecond binding domain binds to the human tumor target antigen and anon-chimpanzee primate tumor target antigen, preferably a macaque tumortarget antigen. It is to be understood, that the second binding domainbinds to at least one non-chimpanzee primate tumor target antigen,however, it may also bind to two, three or more, non-chimpanzee primatetumor target antigen homologs. For example, the second binding domainmay bind to a Cynomogus monkey tumor target antigen and to a Rhesusmonkey tumor target antigen. Preferably, the tumor target antigen isEGFR, CD44v6 or CD30.

For the generation of the second binding domain of the bispecific singlechain antibody molecule of the invention, e.g. bispecific single chainantibodies as defined herein, monoclonal antibodies binding to both ofthe respective human and/or non-chimpanzee primate cell surface tumortarget antigen can be utilized. Appropriate binding domains for thebispecific polypeptide as defined herein e.g. can be derived fromcross-species specific monoclonal antibodies by recombinant methodsdescribed in the art. A monoclonal antibody binding to a human cellsurface antigen and to the homolog of said cell surface antigen in anon-chimpanzee primate can be tested by FACS assays as set forth above.It is evident to those skilled in the art that cross-species specificantibodies can also be generated by hybridoma techniques described inthe literature (Milstein and Köhler, Nature 256 (1975), 495-7). Forexample, mice may be alternately immunized with human and non-chimpanzeeprimate cell surface antigen. From these mice, cross-species specificantibody-producing hybridoma cells are isolated via hybridoma technologyand analysed by FACS as set forth above. The generation and analysis ofbispecific polypeptides such as bispecific single chain antibodiesexhibiting cross-species specificity as described herein is shown in thefollowing examples. The advantages of the bispecific single chainantibodies exhibiting cross-species specificity include the pointsenumerated herein.

In the event that a tylopoda-derived antibody variable domain is used inthe first and/or second portion of a bispecific antibody according tothis embodiment of the invention, said first and/or second portion mayadvantageously be derived independently from camel, llama or/anddromedary. This use of such “camelid” antibodies allows the researcherseeking to develop or optimize bispecific antibodies according to thisembodiment of the invention to capitalize on the unique types ofantibodies known to be produced by these species. These species arenamely known to produce high affinity antibodies of only a singlevariable domain. In the event that a tylopoda antibody is used as thesource for the antibody variable domain in the first and/or secondportion of the bispecific antibody, it is advantageous to use the VHHdomain or a modified variant thereof.

The term “VHH” denotes a variable region of a heavy chain of a so-called“camelid” antibody. Camelid antibodies comprise a heavy chain, but lacka light chain. As such, a VHH region from such a camelid antibodyrepresents the minimal structural element required to specifically bindto an antigen of interest in these species. Camelid VHH domains havebeen found to bind to antigen with high affinity (Desmyter et al. 2001.J Biol Chem 276, 26285-90) and possess high stability in solution (Ewertet al. 2002. Biochemistry 41, 3628-36).

In one embodiment of the invention, the bispecific single chain antibodymolecule of the invention comprises a (first) binding domain consistingof one antibody variable domain capable of binding to an epitope ofhuman and non-chimpanzee primate CD3 epsilon chain and a second bindingdomain consisting of an antibody variable domain capable of binding to ahuman and an non-chimpanzee tumor target antigen. Preferably, the tumortarget antigen is EGFR, CD44v6 or CD30. The first binding domain ispreferably a VH domain or a VHH domain. The second binding domain maycomprise one antibody variable domain (preferably a VH or VHH domain) ortwo antibody variable domains (preferably a scFv, i.e. VH-VL or VL-VH).In a preferred embodiment, at least one of said first or second bindingdomain is CDR-grafted, humanized or human. In another preferredembodiment, at least one of said first or second binding domain is a VHHdomain. Preferably, the antibody variable domain of the first bindingdomain of the bispecific antibody of the invention comprises CDR 1 ofSEQ ID NO: 398, CDR 2 of SEQ ID NO. 399 and CDR 3 of SEQ ID NO. 400.Even more preferred, the first binding domain of the bispecific singlechain antibody molecule comprises an antibody variable domain as shownin SEQ ID NO. 397 or an amino acid sequence at least 80%, more preferredat least 90% or 95% identical, most preferred at least 96% identical tothe amino acid sequence of SEQ ID NO. 397.

In another embodiment of the invention, the bispecific single chainantibody molecule comprises a first binding domain capable of binding toan epitope of the human and non-chimpanzee primate CD3 epsilon chain,wherein the epitope is part of an amino acid sequence comprised in thegroup consisting of SEQ ID NOs. 2, 4, 6, and 8, and a second bindingdomain consisting of one antibody variable domain capable of binding toan epitope of a human and a non-chimpanzee primate tumor target antigen.Preferably, the tumor target antigen is EGFR, CD44v6 or CD30.

In this embodiment, the first binding domain comprises one (preferably aVH domain or a VHH domain) or two variable domains (preferably a scFv,i.e. VH-VL or VL-VH). In a preferred embodiment, at least one of saidfirst or second binding domain is CDR-grafted, humanized or human. Ifthe first binding domain of the bispecific single chain antibodymolecule of the invention comprises two variable domains (VH-VL orVL-VH) it is preferred that the first binding domain capable of bindingto an epitope of human and non-chimpanzee primate CD3ε chain comprises aVL region comprising CDR-L1, CDR-L2 and CDR-L3 selected from:

-   (a) CDR-L1 as depicted in SEQ ID NO. 27, CDR-L2 as depicted in SEQ    ID NO. 28 and CDR-L3 as depicted in SEQ ID NO. 29;-   (b) CDR-L1 as depicted in SEQ ID NO. 117, CDR-L2 as depicted in SEQ    ID NO. 118 and CDR-L3 as depicted in SEQ ID NO. 119; and-   (c) CDR-L1 as depicted in SEQ ID NO. 153, CDR-L2 as depicted in SEQ    ID NO. 154 and CDR-L3 as depicted in SEQ ID NO. 155.

The variable regions, i.e. the variable light chain (“L” or “VL”) andthe variable heavy chain (“H” or “VH”) are understood in the art toprovide the binding domain of an antibody. This variable regions harborthe complementary determining regions. The term “complementarydetermining region” (CDR) is well known in the art to dictate theantigen specificity of an antibody. The term “CDR-L” or “L CDR” or“LCDR” refers to CDRs in the VL, whereas the term “CDR-H” or “H CDR” or“HCDR” refers to the CDRs in the VH.

In an alternatively preferred embodiment of the bispecific single chainantibody molecule of the invention the first binding domain capable ofbinding to an epitope of human and non-chimpanzee primate CD3ε chaincomprises a VH region comprising CDR-H 1, CDR-H2 and CDR-H3 selectedfrom:

-   (a) CDR-H1 as depicted in SEQ ID NO. 12, CDR-H2 as depicted in SEQ    ID NO. 13 and CDR-H3 as depicted in SEQ ID NO. 14;-   (b) CDR-H1 as depicted in SEQ ID NO. 30, CDR-H2 as depicted in SEQ    ID NO. 31 and CDR-H3 as depicted in SEQ ID NO. 32;-   (c) CDR-H1 as depicted in SEQ ID NO. 48, CDR-H2 as depicted in SEQ    ID NO. 49 and CDR-H3 as depicted in SEQ ID NO. 50;-   (d) CDR-H1 as depicted in SEQ ID NO. 66, CDR-H2 as depicted in SEQ    ID NO. 67 and CDR-H3 as depicted in SEQ ID NO. 68;-   (e) CDR-H1 as depicted in SEQ ID NO. 84, CDR-H2 as depicted in SEQ    ID NO. 85 and CDR-H3 as depicted in SEQ ID NO. 86;-   (f) CDR-H1 as depicted in SEQ ID NO. 102, CDR-H2 as depicted in SEQ    ID NO. 103 and CDR-H3 as depicted in SEQ ID NO. 104;-   (g) CDR-H1 as depicted in SEQ ID NO. 120, CDR-H2 as depicted in SEQ    ID NO. 121 and CDR-H3 as depicted in SEQ ID NO. 122;-   (h) CDR-H1 as depicted in SEQ ID NO. 138, CDR-H2 as depicted in SEQ    ID NO. 139 and CDR-H3 as depicted in SEQ ID NO. 140;-   (i) CDR-H1 as depicted in SEQ ID NO. 156, CDR-H2 as depicted in SEQ    ID NO. 157 and CDR-H3 as depicted in SEQ ID NO. 158; and-   (j) CDR-H1 as depicted in SEQ ID NO. 174, CDR-H2 as depicted in SEQ    ID NO. 175 and CDR-H3 as depicted in SEQ ID NO. 176.

It is further preferred that the binding domain capable of binding to anepitope of human and non-chimpanzee primate CD3ε chain comprises a VLregion selected from the group consisting of a VL region as depicted inSEQ ID NO. 35, 39, 125, 129, 161 or 165.

It is alternatively preferred that the first binding domain capable ofbinding to an epitope of human and non-chimpanzee primate CD3ε chaincomprises a VH region selected from the group consisting of a VH regionas depicted in SEQ ID NO. 15, 19, 33, 37, 51, 55, 69, 73, 87, 91, 105,109, 123, 127, 141, 145, 159, 163, 177 or 181.

More preferably, the bispecific single chain antibody molecule of theinvention is characterized by the first binding domain capable ofbinding to an epitope of human and non-chimpanzee primate CD3ε chain,which comprises a VL region and a VH region selected from the groupconsisting of:

-   (a) a VL region as depicted in SEQ ID NO. 17 or 21 and a VH region    as depicted in SEQ ID NO. 15 or 19;-   (b) a VL region as depicted in SEQ ID NO. 35 or 39 and a VH region    as depicted in SEQ ID NO. 33 or 37;-   (c) a VL region as depicted in SEQ ID NO. 53 or 57 and a VH region    as depicted in SEQ ID NO. 51 or 55;-   (d) a VL region as depicted in SEQ ID NO. 71 or 75 and a VH region    as depicted in SEQ ID NO. 69 or 73;-   (e) a VL region as depicted in SEQ ID NO. 89 or 93 and a VH region    as depicted in SEQ ID NO. 87 or 91;-   (f) a VL region as depicted in SEQ ID NO. 107 or 111 and a VH region    as depicted in SEQ ID NO. 105 or 109;-   (g) a VL region as depicted in SEQ ID NO. 125 or 129 and a VH region    as depicted in SEQ ID NO. 123 or 127;-   (h) a VL region as depicted in SEQ ID NO. 143 or 147 and a VH region    as depicted in SEQ ID NO. 141 or 145;-   (i) a VL region as depicted in SEQ ID NO. 161 or 165 and a VH region    as depicted in SEQ ID NO. 159 or 163; and-   (j) a VL region as depicted in SEQ ID NO. 179 or 183 and a VH region    as depicted in SEQ ID NO. 177 or 181.

According to a preferred embodiment of the bispecific single chainantibody molecule of the invention the pairs of VH-regions andVL-regions in the first binding domain binding to CD3 epsilon are in theformat of a single chain antibody (scFv). The VH and VL regions arearranged in the order VH-VL or VL-VH. It is preferred that the VH-regionis positioned N-terminally to a linker sequence. The VL-region ispositioned C-terminally of the linker sequence. Put in other words, thedomain arrangement in the CD3 binding domain of the bispecific singlechain antibody molecule of the invention is preferably VH-VL, with saidCD3 binding domain located C-terminally to the second (cell surfaceantigen) binding domain. Preferably the VH-VL comprises or is SEQ ID NO.185.

A preferred embodiment of the above described bispecific single chainantibody molecule of the invention is characterized by the first bindingdomain capable of binding to an epitope of human and non-chimpanzeeprimate CD3ε chain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97,113, 115, 131, 133, 149, 151, 167, 169, 185 or 187.

According to a preferred embodiment of the invention an abovecharacterized bispecific single chain antibody molecule comprises agroup of the following sequences as CDR1-3 in the second binding domainselected from the group consisting of:

-   -   (a) CDR 1 of SEQ ID NO: 376, CDR 2 of SEQ ID NO. 377 and CDR 3        of SEQ ID NO. 378; and    -   (b) CDR 1 of SEQ ID NO: 387, CDR 2 of SEQ ID NO. 388 and CDR 3        of SEQ ID NO. 389.

The sequences of the corresponding VL- and VH-regions of the secondbinding domain of the bispecific single chain antibody molecule of theinvention as well as of the respective scFvs are shown in the sequencelisting.

In the bispecific single chain antibody molecule of the invention thebinding domains are arranged as exemplified in the appended examples.

A even more preferred embodiment of the invention concerns an abovecharacterized bispecific single chain antibody, wherein the secondbinding domain comprises an antibody variable domain as shown in SEQ IDNO. 375 or 386 or an amino acid sequence at least 80%, more preferred atleast 90% or 95% identical, most preferred at least 96% identical to theamino acid sequence of SEQ ID NO. 375 or 386.

A particularly preferred embodiment of the invention concerns abispecific single chain antibody molecule, wherein the bispecific singlechain antibody molecule comprises a sequence selected from:

-   -   (a) an amino acid sequence as depicted in any of SEQ ID NOs.        380, 382, 384, 391, 393 or 395;    -   (b) an amino acid sequence encoded by a nucleic acid sequence as        depicted in any of SEQ ID NOs: 381, 383, 385, 392, 394 or 396;        and    -   (c) an amino acid sequence at least 90% identical, more        preferred at least 95% identical, most preferred at least 96%        identical to the amino acid sequence of (a) or (b).

The invention relates to a bispecific single chain antibody moleculecomprising an amino acid sequence as depicted in any of SEQ ID NOs: 380,382, 384, 391, 393 or 395, as well as to an amino acid sequences atleast 85% identical, preferably 90%, more preferred at least 95%identical, most preferred at least 96, 97, 98, or 99 identical to theamino acid sequence of SEQ ID NOs: 380, 382, 384, 391, 393 or 395. Theinvention relates also to the corresponding nucleic acid sequences asdepicted in any of SEQ ID NOs: 381, 383, 385, 392, 394, or 396 as wellas to nucleic acid sequences at least 85% identical, preferably 90%,more preferred at least 95 identical, most preferred at least 96, 97,98, or 99% identical to the nucleic acid sequences shown in SEQ ID NOs:381, 383, 385, 392, 394, or 396. It is to be understood that thesequence identity is determined over the entire nucleotide or amino acidsequence. For sequence alignments, for example, the programs Gap orBestFit can be used (Needleman and Wunsch J. Mol. Biol. 48 (1970),443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489), whichis contained in the GCG software package (Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711 (1991). It is a routine methodfor those skilled in the art to determine and identify a nucleotide oramino acid sequence having e.g. 85% (90%, 95%, 96%, 97%, 98% or 99%)sequence identity to the nucleotide or amino acid sequences of thebispecific single single chain antibody of the invention. For example,according to Crick's Wobble hypothesis, the 5′ base on the anti-codon isnot as spatially confined as the other two bases, and could thus havenon-standard base pairing. Put in other words: the third position in acodon triplet may vary so that two triplets which differ in this thirdposition may encode the same amino acid residue. Said hypothesis is wellknown to the person skilled in the art (see e.g.http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J Mol Biol 19(1966): 548-55).

Preferred domain arrangements in the single domain bispecific singlechain antibody constructs of the invention are shown in the followingexamples.

In a preferred embodiment of the invention, the bispecific single chainantibodies are cross-species specific for CD3 epsilon and for human andnon-chimpanzee primate tumor target antigens recognized by their secondbinding domain.

In an alternative embodiment the present invention provides a nucleicacid sequence encoding an above described bispecific single chainantibody molecule of the invention.

The present invention also relates to a vector comprising the nucleicacid molecule of the present invention.

Many suitable vectors are known to those skilled in molecular biology,the choice of which would depend on the function desired and includeplasmids, cosmids, viruses, bacteriophages and other vectors usedconventionally in genetic engineering. Methods which are well known tothose skilled in the art can be used to construct various plasmids andvectors; see, for example, the techniques described in Sambrook et al.(loc cit.) and Ausubel, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y. (1989), (1994).Alternatively, the polynucleotides and vectors of the invention can bereconstituted into liposomes for delivery to target cells. As discussedin further details below, a cloning vector was used to isolateindividual sequences of DNA. Relevant sequences can be transferred intoexpression vectors where expression of a particular polypeptide isrequired. Typical cloning vectors include pBluescript SK, pGEM, pUC9,pBR322 and pGBT9. Typical expression vectors include pTRE, pCAL-n-EK,pESP-1, pOP13CAT.

Preferably said vector comprises a nucleic acid sequence which is aregulatory sequence operably linked to said nucleic acid sequencedefined herein.

The term “regulatory sequence” refers to DNA sequences, which arenecessary to effect the expression of coding sequences to which they areligated. The nature of such control sequences differs depending upon thehost organism. In prokaryotes, control sequences generally includepromoter, ribosomal binding site, and terminators. In eukaryotesgenerally control sequences include promoters, terminators and, in someinstances, enhancers, transactivators or transcription factors. The term“control sequence” is intended to include, at a minimum, all componentsthe presence of which are necessary for expression, and may also includeadditional advantageous components.

The term “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. A control sequence “operably linked”to a coding sequence is ligated in such a way that expression of thecoding sequence is achieved under conditions compatible with the controlsequences. In case the control sequence is a promoter, it is obvious fora skilled person that double-stranded nucleic acid is preferably used.

Thus, the recited vector is preferably an expression vector. An“expression vector” is a construct that can be used to transform aselected host and provides for expression of a coding sequence in theselected host. Expression vectors can for instance be cloning vectors,binary vectors or integrating vectors. Expression comprisestranscription of the nucleic acid molecule preferably into atranslatable mRNA. Regulatory elements ensuring expression inprokaryotes and/or eukaryotic cells are well known to those skilled inthe art. In the case of eukaryotic cells they comprise normallypromoters ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Possible regulatory elements permitting expression inprokaryotic host cells comprise, e.g., the P_(L), lac, trp or tacpromoter in E. coli, and examples of regulatory elements permittingexpression in eukaryotic host cells are the AOX1 or GAL1 promoter inyeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells.

Beside elements, which are responsible for the initiation oftranscription such regulatory elements may also comprise transcriptiontermination signals, such as the SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. Furthermore, depending on theexpression system used leader sequences capable of directing thepolypeptide to a cellular compartment or secreting it into the mediummay be added to the coding sequence of the recited nucleic acid sequenceand are well known in the art; see also the appended Examples. Theleader sequence(s) is (are) assembled in appropriate phase withtranslation, initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated protein, ora portion thereof, into the periplasmic space or extracellular medium.Optionally, the heterologous sequence can encode a fusion proteinincluding an N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product; see supra. In this context, suitableexpression vectors are known in the art such as Okayama-Berg cDNAexpression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3(In-vitrogene), pEF-DHFR, pEF-ADA or pEF-neo (Mack et al. PNAS (1995)92, 7021-7025 and Raum et al. Cancer Immunol Immunother (2001) 50(3),141-150) or pSPORT1 (GIBCO BRL).

Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming of transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused. Once the vector has been incorporated into the appropriate host,the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and as desired, the collectionand purification of the bispecific single chain antibody molecule of theinvention may follow; see, e.g., the appended examples.

An alternative expression system, which can be used to express a cellcycle interacting protein is an insect system. In one such system,Autographa californica nuclear polyhedrosis virus (AcNPV) is used as avector to express foreign genes in Spodoptera frugiperda cells or inTrichoplusia larvae. The coding sequence of a recited nucleic acidmolecule may be cloned into a nonessential region of the virus, such asthe polyhedrin gene, and placed under control of the polyhedrinpromoter. Successful insertion of said coding sequence will render thepolyhedrin gene inactive and produce recombinant virus lacking coatprotein coat. The recombinant viruses are then used to infect S.frugiperda cells or Trichoplusia larvae in which the protein of theinvention is expressed (Smith, J. Virol. 46 (1983), 584; Engelhard,Proc. Nat. Acad. Sci. USA 91 (1994), 3224-3227).

Additional regulatory elements may include transcriptional as well astranslational enhancers. Advantageously, the above-described vectors ofthe invention comprise a selectable and/or scorable marker.

Selectable marker genes useful for the selection of transformed cellsand, e.g., plant tissue and plants are well known to those skilled inthe art and comprise, for example, antimetabolite resistance as thebasis of selection for dhfr, which confers resistance to methotrexate(Reiss, Plant Physiol. (Life Sci. Adv.) 13 (1994), 143-149); npt, whichconfers resistance to the aminoglycosides neomycin, kanamycin andparomycin (Herrera-Estrella, EMBO J. 2 (1983), 987-995) and hygro, whichconfers resistance to hygromycin (Marsh, Gene 32 (1984), 481-485).Additional selectable genes have been described, namely trpB, whichallows cells to utilize indole in place of tryptophan; hisD, whichallows cells to utilize histinol in place of histidine (Hartman, Proc.Natl. Acad. Sci. USA 85 (1988), 8047); mannose-6-phosphate isomerasewhich allows cells to utilize mannose (WO 94/20627) and ODC (ornithinedecarboxylase) which confers resistance to the ornithine decarboxylaseinhibitor, 2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, 1987, In:Current Communications in Molecular Biology, Cold Spring HarborLaboratory ed.) or deaminase from Aspergillus terreus which confersresistance to Blasticidin S (Tamura, Biosci. Biotechnol. Biochem. 59(1995), 2336-2338).

Useful scorable markers are also known to those skilled in the art andare commercially available. Advantageously, said marker is a geneencoding luciferase (Giacomin, Pl. Sci. 116 (1996), 59-72; Scikantha, J.Bact. 178 (1996), 121), green fluorescent protein (Gerdes, FEBS Lett.389 (1996), 44-47) or β-glucuronidase (Jefferson, EMBO J. 6 (1987),3901-3907). This embodiment is particularly useful for simple and rapidscreening of cells, tissues and organisms containing a recited vector.

As described above, the recited nucleic acid molecule can be used aloneor as part of a vector to express the bispecific single chain antibodymolecule of the invention in cells, for, e.g., purification but also forgene therapy purposes. The nucleic acid molecules or vectors containingthe DNA sequence(s) encoding any one of the above described bispecificsingle chain antibody molecule of the invention is introduced into thecells which in turn produce the polypeptide of interest. Gene therapy,which is based on introducing therapeutic genes into cells by ex-vivo orin-vivo techniques is one of the most important applications of genetransfer. Suitable vectors, methods or gene-delivery systems forin-vitro or in-vivo gene therapy are described in the literature and areknown to the person skilled in the art; see, e.g., Giordano, NatureMedicine 2 (1996), 534-539; Schaper, Circ. Res. 79 (1996), 911-919;Anderson, Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239;Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77 (1995),1077-1086; Onodera, Blood 91 (1998), 30-36; Verma, Gene Ther. 5 (1998),692-699; Nabel, Ann. N.Y. Acad. Sci. 811 (1997), 289-292; Verzeletti,Hum. Gene Ther. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996),714-716; WO 94/29469; WO 97/00957, U.S. Pat. No. 5,580,859; U.S. Pat.No. 5,589,466; or Schaper, Current Opinion in Biotechnology 7 (1996),635-640; dos Santos Coura and Nardi Virol J. (2007), 4:99. The recitednucleic acid molecules and vectors may be designed for directintroduction or for introduction via liposomes, or viral vectors (e.g.,adenoviral, retroviral) into the cell. Preferably, said cell is a germline cell, embryonic cell, or egg cell or derived there from, mostpreferably said cell is a stem cell. An example for an embryonic stemcell can be, inter alia, a stem cell as described in Nagy, Proc. Natl.Acad. Sci. USA 90 (1993), 8424-8428.

The invention also provides for a host transformed or transfected with avector of the invention. Said host may be produced by introducing theabove described vector of the invention or the above described nucleicacid molecule of the invention into the host. The presence of at leastone vector or at least one nucleic acid molecule in the host may mediatethe expression of a gene encoding the above described single chainantibody constructs.

The described nucleic acid molecule or vector of the invention, which isintroduced in the host may either integrate into the genome of the hostor it may be maintained extrachromosomally.

The host can be any prokaryote or eukaryotic cell.

The term “prokaryote” is meant to include all bacteria, which can betransformed or transfected with DNA or RNA molecules for the expressionof a protein of the invention. Prokaryotic hosts may include gramnegative as well as gram positive bacteria such as, for example, E.coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. Theterm “eukaryotic” is meant to include yeast, higher plant, insect andpreferably mammalian cells. Depending upon the host employed in arecombinant production procedure, the protein encoded by thepolynucleotide of the present invention may be glycosylated or may benon-glycosylated. Especially preferred is the use of a plasmid or avirus containing the coding sequence of the bispecific single chainantibody molecule of the invention and genetically fused thereto anN-terminal FLAG-tag and/or C-terminal His-tag. Preferably, the length ofsaid FLAG-tag is about 4 to 8 amino acids, most preferably 8 aminoacids. An above described polynucleotide can be used to transform ortransfect the host using any of the techniques commonly known to thoseof ordinary skill in the art. Furthermore, methods for preparing fused,operably linked genes and expressing them in, e.g., mammalian cells andbacteria are well-known in the art (Sambrook, loc cit.).

Preferably, said the host is a bacterium or an insect, fungal, plant oranimal cell.

It is particularly envisaged that the recited host may be a mammaliancell. Particularly preferred host cells comprise CHO cells, COS cells,myeloma cell lines like SP2/0 or NS/0. As illustrated in the appendedexamples, particularly preferred are CHO-cells as hosts.

More preferably said host cell is a human cell or human cell line, e.g.per.c6 (Kroos, Biotechnol. Prog., 2003, 19:163-168).

In a further embodiment, the present invention thus relates to a processfor the production of a bispecific single chain antibody molecule of theinvention, said process comprising culturing a host of the inventionunder conditions allowing the expression of the bispecific single chainantibody molecule of the invention and recovering the producedpolypeptide from the culture.

The transformed hosts can be grown in fermentors and cultured accordingto techniques known in the art to achieve optimal cell growth. Thebispecific single chain antibody molecule of the invention can then beisolated from the growth medium, cellular lysates, or cellular membranefractions. The isolation and purification of the, e.g., microbiallyexpressed bispecific single chain antibody molecules may be by anyconventional means such as, for example, preparative chromatographicseparations and immunological separations such as those involving theuse of monoclonal or polyclonal antibodies directed, e.g., against a tagof the bispecific single chain antibody molecule of the invention or asdescribed in the appended examples.

The conditions for the culturing of a host, which allow the expressionare known in the art to depend on the host system and the expressionsystem/vector used in such process. The parameters to be modified inorder to achieve conditions allowing the expression of a recombinantpolypeptide are known in the art. Thus, suitable conditions can bedetermined by the person skilled in the art in the absence of furtherinventive input.

Once expressed, the bispecific single chain antibody molecule of theinvention can be purified according to standard procedures of the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like; see, Scopes, “ProteinPurification”, Springer-Verlag, N.Y. (1982). Substantially purepolypeptides of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity are most preferred, for pharmaceuticaluses. Once purified, partially or to homogeneity as desired, thebispecific single chain antibody molecule of the invention may then beused therapeutically (including extracorporeally) or in developing andperforming assay procedures. Furthermore, examples for methods for therecovery of the bispecific single chain antibody molecule of theinvention from a culture are described in detail in the appendedexamples. The recovery can also be achieved by a method for theisolation of the bispecific single chain antibody molecule of theinvention capable of binding to an epitope of human and non-chimpanzeeprimate CD3 epsilon (CD3

, the method comprising the steps of:

(a) contacting the polypeptide(s) with an N-terminal fragment of theextracellular domain of CD3ε of maximal 27 amino acids comprising theamino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQ ID NO. 341) orGln-Asp-Gly-Asn-Glu-Glu-Ile-Gly (SEQ ID NO. 342), fixed via itsC-terminus to a solid phase;(b) eluting the bound polypeptide(s) from said fragment; and(c) isolating the polypeptide(s) from the eluate of (b).

It is preferred that the polypeptide(s) isolated by the above method ofthe invention are human.

This method or the isolation of the bispecific single chain antibodymolecule of the invention is understood as a method for the isolation ofone or more different polypeptides with the same specificity for thefragment of the extracellular domain of CD3ε comprising at itsN-terminus the amino acid sequence Gln-Asp-Gly-Asn-Glu-Glu-Met-Gly (SEQID NO. 341) or Gln-Asp-Gly-Asn-Glu-Glu-Ile-Gly (SEQ ID NO. 342) from aplurality of polypeptide candidates as well as a method for thepurification of a polypeptide from a solution. A non-limiting examplefor the latter method for the purification of a bispecific single chainantibody molecule from a solution is e.g. the purification of arecombinantly expressed bispecific single chain antibody molecule from aculture supernatant or a preparation from such culture. As stated abovethe fragment used in this method is an N-terminal fragment of theextracellular domain of the primate CD3ε molecule. The amino acidsequence of the extracellular domain of the CD3ε molecule of differentspecies is depicted in SEQ ID NOs: 1, 3, 5 and 7. The two forms of theN-terminal octamer are depicted in SEQ ID NOs: 341 and 342. It ispreferred that this N-terminus is freely available for binding of thepolypeptides to be identified by the method of the invention. The term“freely available” is understood in the context of the invention as freeof additional motives such as a His-tag. The interference of such aHis-tag with a binding molecule identified by the method of theinvention is described in the appended Examples 6 and 20.

According to this method said fragment is fixed via its C-terminus to asolid phase. The person skilled in the art will easily and without anyinventive ado elect a suitable solid phase support dependent from theused embodiment of the method of the invention. Examples for a solidsupport comprise but are not limited to matrices like beads (e.g.agarose beads, sepharose beads, polystyrol beads, dextran beads), plates(culture plates or MultiWell plates) as well as chips known e.g. fromBiacore®. The selection of the means and methods for thefixation/immobilization of the fragment to said solid support depend onthe election of the solid support. A commonly used method for thefixation/immobilization is a coupling via an N-hydroxysuccinimide (NHS)ester. The chemistry underlying this coupling as well as alternativemethods for the fixation/immobilization are known to the person skilledin the art, e.g. from Hermanson “Bioconjugate Techniques”, AcademicPress, Inc. (1996). For the fixation to/immobilization onchromatographic supports the following means are commonly used:NHS-activated sepharose (e.g. HiTrap-NHS of GE Life Science-Amersham),CnBr-activated sepharose (e.g. GE Life Science-Amersham), NHS-activateddextran beads (Sigma) or activated polymethacrylate. These reagents mayalso be used in a batch approach. Moreover, dextran beads comprisingiron oxide (e.g. available from Miltenyi) may be used in a batchapproach. These beads may be used in combination with a magnet for theseparation of the beads from a solution. Polypeptides can be immobilizedon a Biacore chip (e.g. CM5 chips) by the use of NHS activatedcarboxymethyldextran. Further examples for an appropriate solid supportare amine reactive MultiWell plates (e.g. Nunc Immobilizer™ plates).According to this method said fragment of the extracellular domain ofCD3 epsilon can be directly coupled to the solid support or via astretch of amino acids, which might be a linker or anotherprotein/polypeptide moiety. Alternatively, the extracellular domain ofCD3 epsilon can be indirectly coupled via one or more adaptormolecule(s).

Means and methods for the eluation of a peptide or polypeptide bound toan immobilized epitope are well known in the art. The same holds truefor methods for the isolation of the identified polypeptide(s) from theeluate.

A method for the isolation of one or more different bispecific singlechain antibody molecule(s) with the same specificity for the fragment ofthe extracellular domain of CD3ε comprising at its N-terminus the aminoacid sequence Gln-Asp-Gly-Asn-Glu-Glu-X-Gly (with X being Met or Ile)from a plurality of polypeptide candidates may comprise one or moresteps of the following methods for the selection of antigen-specificentities:

CD3ε specific binding domains can be selected from antibody derivedrepertoires. A phage display library can be constructed based onstandard procedures, as for example disclosed in “Phage Display: ALaboratory Manual”; Ed. Barbas, Burton, Scott & Silverman; Cold SpringHarbor Laboratory Press, 2001. The format of the antibody fragments inthe antibody library can be scFv, but may generally also be a Fabfragment or even a single domain antibody fragment. For the isolation ofantibody fragments naïve antibody fragment libraries may be used. Forthe selection of potentially low immunogenic binding entities in latertherapeutic use, human antibody fragment libraries may be favourable forthe direct selection of human antibody fragments. In some cases they mayform the basis for synthetic antibody libraries (Knappik et al. J Mol.Biol. 2000, 296:57 ff). The corresponding format may be Fab, scFv (asdescribed below) or domain antibodies (dAbs, as reviewed in Holt et al.,Trends Biotechnol. 2003, 21:484 ff).

It is also known in the art that in many cases there is no immune humanantibody source available against the tumor target antigen. Thereforeanimals are immunized with the tumor target antigen and the respectiveantibody libraries isolated from animal tissue as e.g. spleen or PBMCs.The N-terminal fragment may be biotinylated or covalently linked toproteins like KLH or bovine serum albumin (BSA). According to commonapproaches rodents are used for immunization. Some immune antibodyrepertoires of non-human origin may be especially favourable for otherreasons, e.g. for the presence of single domain antibodies (VHH) derivedfrom cameloid species (as described in Muyldermans, J Biotechnol.74:277; De Genst et al. Dev Como Immunol. 2006, 30:187 ff). Therefore acorresponding format of the antibody library may be Fab, scFv (asdescribed below) or single domain antibodies (VHH).

In one possible approach ten weeks old F1 mice from balb/c×C57blackcrossings can be immunized with whole cells e.g. expressingtransmembrane EpCAM N-terminally displaying as translational fusion theN-terminal amino acids 1 to 27 of the mature CD3ε chain. Alternatively,mice can be immunized with 1-27 CD3 epsilon-Fc fusion protein (acorresponding approach is described in the appended Example 2). Afterbooster immunization(s), blood samples can be taken and antibody serumtiter against the CD3-positive T cells can be tested e.g. in FACSanalysis. Usually, serum titers are significantly higher in immunizedthan in non-immunized animals.

Immunized animals may form the basis for the construction of immuneantibody libraries. Examples of such libraries comprise phage displaylibraries. Such libraries may be generally constructed based on standardprocedures, as for example disclosed in “Phage Display: A LaboratoryManual”; Ed. Barbas, Burton, Scott & Silverman; Cold Spring HarborLaboratory Press, 2001.

The non-human antibodies can also be humanized via phage display due tothe generation of more variable antibody libraries that can besubsequently enriched for binders during selection.

In a phage display approach any one of the pools of phages that displaysthe antibody libraries forms a basis to select binding entities usingthe respective antigen as target molecule. The central step in whichantigen specific, antigen bound phages are isolated is designated aspanning. Due to the display of the antibody fragments on the surface ofthe phages, this general method is called phage display. One preferredmethod of selection is the use of small proteins such as the filamentousphage N2 domain translationally fused to the N-terminus of the scFvdisplayed by the phage. Another display method known in the art, whichmay be used to isolate binding entities is the ribosome display method(reviewed in Groves & Osbourn, Expert Opin Biol Ther. 2005, 5:125 ff;Lipovsek & Pluckthun, J Immunol Methods 2004, 290:52 ff). In order todemonstrate binding of scFv phage particles to a 1-27 CD3ε-Fc fusionprotein a phage library carrying the cloned scFv-repertoire can beharvested from the respective culture supernatant by PEG(polyethyleneglycole). ScFv phage particles may be incubated withimmobilized CD3ε Fc fusion protein. The immobilized CD3ε Fc fusionprotein may be coated to a solid phase. Binding entities can be elutedand the eluate can be used for infection of fresh uninfected bacterialhosts. Bacterial hosts successfully transduced with a phagemid copy,encoding a human scFv-fragment, can be selected again for carbenicillinresistance and subsequently infected with e.g. VCMS 13 helper phage tostart the second round of antibody display and in vitro selection. Atotal of 4 to 5 rounds of selections is carried out, normally. Thebinding of isolated binding entities can be tested on CD3 epsilonpositive Jurkat cells, HPBall cells, PBMCs or transfected eukaryoticcells that carry the N-terminal CD3ε sequence fused to surface displayedEpCAM using a flow cytometric assay (see appended Example 4).

Preferably, the above method may be a method, wherein the fragment ofthe extracellular domain of CD3ε consists of one or more fragments of apolypeptide having an amino acid sequence of any one depicted in SEQ IDNOs. 2, 4, 6 or 8. More preferably, said fragment is 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acidresidues in length.

This method of identification of a bispecific single chain antibodymolecule may be a method of screening a plurality of bispecific singlechain antibody molecules comprising a cross-species specific bindingdomain binding to an epitope of human and non-chimpanzee primate CD3ε.Alternatively, the method of identification is a method ofpurification/isolation of a bispecific single chain antibody moleculecomprising a cross-species specific binding domain binding to an epitopeof human and non-chimpanzee primate CD3ε.

Furthermore, the invention provides for a composition comprising abispecific single chain antibody molecule of the invention or abispecific single chain antibody as produced by the process disclosedabove. Preferably, said composition is a pharmaceutical composition.

The invention provides also for a bispecific single chain antibodymolecule as defined herein, or produced according to the process asdefined herein, wherein said bispecific single chain antibody moleculeis for use in the prevention, treatment or amelioration of cancer.Preferably, CD44v6×CD3 bispecific single chain antibodies of theinvention can be used as therapeutic agents in order to treat squamouscell carcinoma. In particular, said CD44v6×CD3 bispecific single chainantibodies are useful to kill the migratory subset of colorectalcarcinoma cells. Preferably, EGFR×CD3 bispecific single chain antibodiescan be used as therapeutic agents in order to treat epithelial cancer.Preferably, CD30×CD3 bispecific single chain antibodies can be used astherapeutic agents in order to treat Hodgkin's lymphoma. It is preferredthat the bispecific single chain is further comprising suitableformulations of carriers, stabilizers and/or excipients. Moreover, it ispreferred that said bispecific single chain antibody molecule issuitable to be administered in combination with an additional drug. Saiddrug may be a non-proteinaceous compound or a proteinaceous compound andmay be administered simultaneously or non-simultaneously with thebispecific single chain antibody molecule as defined herein.

In accordance with the invention, the term “pharmaceutical composition”relates to a composition for administration to a patient, preferably ahuman patient. The particular preferred pharmaceutical composition ofthis invention comprises bispecific single chain antibodies directedagainst and generated against context-independent CD3 epitopes.Preferably, the pharmaceutical composition comprises suitableformulations of carriers, stabilizers and/or excipients. In a preferredembodiment, the pharmaceutical composition comprises a composition forparenteral, transdermal, intraluminal, intraarterial, intrathecal and/orintranasal administration or by direct injection into tissue. It is inparticular envisaged that said composition is administered to a patientvia infusion or injection. Administration of the suitable compositionsmay be effected by different ways, e.g., by intravenous,intraperitoneal, subcutaneous, intramuscular, topical or intradermaladministration. In particular, the present invention provides for anuninterrupted administration of the suitable composition. As anon-limiting example, uninterrupted, i.e. continuous administration maybe realized by a small pump system worn by the patient for metering theinflux of therapeutic agent into the body of the patient. Thepharmaceutical composition comprising the bispecific single chainantibodies directed against and generated against context-independentCD3 epitopes of the invention can be administered by using said pumpsystems. Such pump systems are generally known in the art, and commonlyrely on periodic exchange of cartridges containing the therapeutic agentto be infused. When exchanging the cartridge in such a pump system, atemporary interruption of the otherwise uninterrupted flow oftherapeutic agent into the body of the patient may ensue. In such acase, the phase of administration prior to cartridge replacement and thephase of administration following cartridge replacement would still beconsidered within the meaning of the pharmaceutical means and methods ofthe invention together make up one “uninterrupted administration” ofsuch therapeutic agent. The continuous or uninterrupted administrationof these bispecific single chain antibodies directed against andgenerated against context-independent CD3 epitopes of this invention maybe intravenuous or subcutaneous by way of a fluid delivery device orsmall pump system including a fluid driving mechanism for driving fluidout of a reservoir and an actuating mechanism for actuating the drivingmechanism. Pump systems for subcutaneous administration may include aneedle or a cannula for penetrating the skin of a patient and deliveringthe suitable composition into the patient's body. Said pump systems maybe directly fixed or attached to the skin of the patient independentlyof a vein, artery or blood vessel, thereby allowing a direct contactbetween the pump system and the skin of the patient. The pump system canbe attached to the skin of the patient for 24 hours up to several days.The pump system may be of small size with a reservoir for small volumes.As a non-limiting example, the volume of the reservoir for the suitablepharmaceutical composition to be administered can be between 0.1 and 50ml.

The continuous administration may be transdermal by way of a patch wornon the skin and replaced at intervals. One of skill in the art is awareof patch systems for drug delivery suitable for this purpose. It is ofnote that transdermal administration is especially amenable touninterrupted administration, as exchange of a first exhausted patch canadvantageously be accomplished simultaneously with the placement of anew, second patch, for example on the surface of the skin immediatelyadjacent to the first exhausted patch and immediately prior to removalof the first exhausted patch. Issues of flow interruption or power cellfailure do not arise.

The composition of the present invention, comprising in particularbispecific single chain antibodies directed against and generatedagainst context-independent CD3 epitopes may further comprise apharmaceutically acceptable carrier. Examples of suitable pharmaceuticalcarriers are well known in the art and include solutions, e.g. phosphatebuffered saline solutions, water, emulsions, such as oil/wateremulsions, various types of wetting agents, sterile solutions,liposomes, etc. Compositions comprising such carriers can be formulatedby well known conventional methods. Formulations can comprisecarbohydrates, buffer solutions, amino acids and/or surfactants.Carbohydrates may be non-reducing sugars, preferably trehalose, sucrose,octasulfate, sorbitol or xylitol. Such formulations may be used forcontinuous administrations which may be intravenuous or subcutaneouswith and/or without pump systems. Amino acids may be charged aminoacids, preferably lysine, lysine acetate, arginine, glutamate and/orhistidine. Surfactants may be detergents, preferably with a molecularweight of >1.2 KD and/or a polyether, preferably with a molecular weightof >3 KD. Non-limiting examples for preferred detergents are Tween 20,Tween 40, Tween 60, Tween 80 or Tween 85. Non-limiting examples forpreferred polyethers are PEG 3000, PEG 3350, PEG 4000 or PEG 5000.Buffer systems used in the present invention can have a preferred pH of5-9 and may comprise citrate, succinate, phosphate, histidine andacetate. The compositions of the present invention can be administeredto the subject at a suitable dose which can be determined e.g. by doseescalating studies by administration of increasing doses of thebispecific single chain antibody molecule of the invention exhibitingcross-species specificity described herein to non-chimpanzee primates,for instance macaques. As set forth above, the bispecific single chainantibody molecule of the invention exhibiting cross-species specificitydescribed herein can be advantageously used in identical form inpreclinical testing in non-chimpanzee primates and as drug in humans.These compositions can also be administered in combination with otherproteinaceous and non-proteinaceous drugs. These drugs may beadministered simultaneously with the composition comprising thebispecific single chain antibody molecule of the invention as definedherein or separately before or after administration of said polypeptidein timely defined intervals and doses. The dosage regimen will bedetermined by the attending physician and clinical factors. As is wellknown in the medical arts, dosages for any one patient depend upon manyfactors, including the patient's size, body surface area, age, theparticular compound to be administered, sex, time and route ofadministration, general health, and other drugs being administeredconcurrently. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, inertgases and the like. In addition, the composition of the presentinvention might comprise proteinaceous carriers, like, e.g., serumalbumin or immunoglobulin, preferably of human origin. It is envisagedthat the composition of the invention might comprise, in addition to thebispecific single chain antibody molecule of the invention definedherein, further biologically active agents, depending on the intendeduse of the composition. Such agents might be drugs acting on thegastro-intestinal system, drugs acting as cytostatica, drugs preventinghyperurikemia, drugs inhibiting immunoreactions (e.g. corticosteroids),drugs modulating the inflammatory response, drugs acting on thecirculatory system and/or agents such as cytokines known in the art.

The biological activity of the pharmaceutical composition defined hereincan be determined for instance by cytotoxicity assays, as described inthe following examples, in WO 99/54440 or by Schlereth et al. (CancerImmunol. Immunother. 20 (2005), 1-12). “Efficacy” or “in vivo efficacy”as used herein refers to the response to therapy by the pharmaceuticalcomposition of the invention, using e.g. standardized NCI responsecriteria. The success or in vivo efficacy of the therapy using apharmaceutical composition of the invention refers to the effectivenessof the composition for its intended purpose, i.e. the ability of thecomposition to cause its desired effect, i.e. depletion of pathologiccells, e.g. tumor cells. The in vivo efficacy may be monitored byestablished standard methods for the respective disease entitiesincluding, but not limited to white blood cell counts, differentials,Fluorescence Activated Cell Sorting, bone marrow aspiration. Inaddition, various disease specific clinical chemistry parameters andother established standard methods may be used. Furthermore,computer-aided tomography, X-ray, nuclear magnetic resonance tomography(e.g. for National Cancer Institute-criteria based response assessment[Cheson B D, Horning S J, Coiffier B, Shipp M A, Fisher R I, Connors JM, Lister T A, Vose J, Grillo-Lopez A, Hagenbeek A, Cabanillas F,Klippensten D, Hiddemann W, Castellino R, Harris N L, Armitage J O,Carter W, Hoppe R, Canellos G P. Report of an international workshop tostandardize response criteria for non-Hodgkin's lymphomas. NCI SponsoredInternational Working Group. J Clin Oncol. 1999 April; 17(4):1244]),positron-emission tomography scanning, white blood cell counts,differentials, Fluorescence Activated Cell Sorting, bone marrowaspiration, lymph node biopsies/histologies, and various cancer specificclinical chemistry parameters (e.g. lactate dehydrogenase) and otherestablished standard methods may be used.

Another major challenge in the development of drugs such as thepharmaceutical composition of the invention is the predictablemodulation of pharmacokinetic properties. To this end, a pharmacokineticprofile of the drug candidate, i.e. a profile of the pharmacokineticparameters that effect the ability of a particular drug to treat a givencondition, is established. Pharmacokinetic parameters of the druginfluencing the ability of a drug for treating a certain disease entityinclude, but are not limited to: half-life, volume of distribution,hepatic first-pass metabolism and the degree of blood serum binding. Theefficacy of a given drug agent can be influenced by each of theparameters mentioned above.

“Half-life” means the time where 50% of an administered drug areeliminated through biological processes, e.g. metabolism, excretion,etc.

By “hepatic first-pass metabolism” is meant the propensity of a drug tobe metabolized upon first contact with the liver, i.e. during its firstpass through the liver. “Volume of distribution” means the degree ofretention of a drug throughout the various compartments of the body,like e.g. intracellular and extracellular spaces, tissues and organs,etc. and the distribution of the drug within these compartments. “Degreeof blood serum binding” means the propensity of a drug to interact withand bind to blood serum proteins, such as albumin, leading to areduction or loss of biological activity of the drug.

Pharmacokinetic parameters also include bioavailability, lag time(Tlag), Tmax, absorption rates, more onset and/or Cmax for a givenamount of drug administered. “Bioavailability” means the amount of adrug in the blood compartment.

“Lag time” means the time delay between the administration of the drugand its detection and measurability in blood or plasma.

“Tmax” is the time after which maximal blood concentration of the drugis reached, and “Cmax” is the blood concentration maximally obtainedwith a given drug. The time to reach a blood or tissue concentration ofthe drug which is required for its biological effect is influenced byall parameters. Pharmacokinetik parameters of bispecific single chainantibodies exhibiting cross-species specificity, which may be determinedin preclinical animal testing in non-chimpanzee primates as outlinedabove are also set forth e.g. in the publication by Schlereth et al.(Cancer Immunol. Immunother. 20 (2005), 1-12).

The term “toxicity” as used herein refers to the toxic effects of a drugmanifested in adverse events or severe adverse events. These side eventsmight refer to a lack of tolerability of the drug in general and/or alack of local tolerance after administration. Toxicity could alsoinclude teratogenic or carcinogenic effects caused by the drug.

The term “safety”, “in vivo safety” or “tolerability” as used hereindefines the administration of a drug without inducing severe adverseevents directly after administration (local tolerance) and during alonger period of application of the drug. “Safety”, “in vivo safety” or“tolerability” can be evaluated e.g. at regular intervals during thetreatment and follow-up period. Measurements include clinicalevaluation, e.g. organ manifestations, and screening of laboratoryabnormalities. Clinical evaluation may be carried out and deviating tonormal findings recorded/coded according to NCI-CTC and/or MedDRAstandards. Organ manifestations may include criteria such asallergy/immunology, blood/bone marrow, cardiac arrhythmia, coagulationand the like, as set forth e.g. in the Common Terminology Criteria foradverse events v3.0 (CTCAE). Laboratory parameters which may be testedinclude for instance haematology, clinical chemistry, coagulationprofile and urine analysis and examination of other body fluids such asserum, plasma, lymphoid or spinal fluid, liquor and the like. Safety canthus be assessed e.g. by physical examination, imaging techniques (i.e.ultrasound, x-ray, CT scans, Magnetic Resonance Imaging (MRI), othermeasures with technical devices (i.e. electrocardiogram), vital signs,by measuring laboratory parameters and recording adverse events. Forexample, adverse events in non-chimpanzee primates in the uses andmethods according to the invention may be examined by histopathologicaland/or histochemical methods.

The term “effective and non-toxic dose” as used herein refers to atolerable dose of the bispecific single chain antibody as defined hereinwhich is high enough to cause depletion of pathologic cells, tumorelimination, tumor shrinkage or stabilization of disease without oressentially without major toxic effects. Such effective and non-toxicdoses may be determined e.g. by dose escalation studies described in theart and should be below the dose inducing severe adverse side events(dose limiting toxicity, DLT).

The above terms are also referred to e.g. in the Preclinical safetyevaluation of biotechnology-derived pharmaceuticals S6; ICH HarmonisedTripartite Guideline; ICH Steering Committee meeting on Jul. 16, 1997.

Moreover, the invention relates to a pharmaceutical compositioncomprising a bispecific single chain antibody molecule of this inventionor produced according to the process according to the invention for theprevention, treatment or amelioration of cancer. Preferably, saidpharmaceutical composition further comprises suitable formulations ofcarriers, stabilizers and/or excipients.

A further aspect of the invention relates to a use of a bispecificsingle chain antibody molecule/polypeptide as defined herein above orproduced according to a process defined herein above, for thepreparation of a pharmaceutical composition for the prevention,treatment or amelioration of a disease. Preferably, said disease iscancer.

In another preferred embodiment of use of the bispecific single chainantibody molecule of the invention said pharmaceutical composition issuitable to be administered in combination with an additional drug, i.e.as part of a co-therapy. In said co-therapy, an active agent may beoptionally included in the same pharmaceutical composition as thebispecific single chain antibody molecule of the invention, or may beincluded in a separate pharmaceutical composition. In this latter case,said separate pharmaceutical composition is suitable for administrationprior to, simultaneously as or following administration of saidpharmaceutical composition comprising the bispecific single chainantibody molecule of the invention. The additional drug orpharmaceutical composition may be a non-proteinaceous compound or aproteinaceous compound. In the case that the additional drug is aproteinaceous compound, it is advantageous that the proteinaceouscompound be capable of providing an activation signal for immuneeffector cells.

Preferably, said proteinaceous compound or non-proteinaceous compoundmay be administered simultaneously or non-simultaneously with thebispecific single chain antibody molecule of the invention, a nucleicacid molecule as defined hereinabove, a vector as defined as definedhereinabove, or a host as defined as defined hereinabove.

Another aspect of the invention relates to a method for the prevention,treatment or amelioration of a disease in a subject in the need thereof,said method comprising the step of administration of an effective amountof a pharmaceutical composition of the invention. Preferably, saiddisease is cancer.

In another preferred embodiment of the method of the invention saidpharmaceutical composition is suitable to be administered in combinationwith an additional drug, i.e. as part of a co-therapy. In saidco-therapy, an active agent may be optionally included in the samepharmaceutical composition as the bispecific single chain antibodymolecule of the invention, or may be included in a separatepharmaceutical composition. In this latter case, said separatepharmaceutical composition is suitable for administration prior to,simultaneously as or following administration of said pharmaceuticalcomposition comprising the bispecific single chain antibody molecule ofthe invention. The additional drug or pharmaceutical composition may bea non-proteinaceous compound or a proteinaceous compound. In the casethat the additional drug is a proteinaceous compound, it is advantageousthat the proteinaceous compound be capable of providing an activationsignal for immune effector cells.

Preferably, said proteinaceous compound or non-proteinaceous compoundmay be administered simultaneously or non-simultaneously with thebispecific single chain antibody molecule of the invention, a nucleicacid molecule as defined hereinabove, a vector as defined as definedhereinabove, or a host as defined as defined hereinabove.

It is preferred for the above described method of the invention thatsaid subject is a human.

In a further aspect, the invention relates to a kit comprising abispecific single chain antibody molecule of the invention, a nucleicacid molecule of the invention, a vector of the invention, or a host ofthe invention.

These and other embodiments are disclosed and encompassed by thedescription and Examples of the present invention. Recombinanttechniques and methods in immunology are described e.g. in Sambrook etal. Molecular Cloning: A Laboratory Manual; Cold Spring HarborLaboratory Press, 3^(rd) edition 2001; Lefkovits; Immunology MethodsManual; The Comprehensive Sourcebook of Techniques; Academic Press,1997; Golemis; Protein-Protein Interactions: A Molecular Cloning Manual;Cold Spring Laboratory Press, 2002. Further literature concerning anyone of the antibodies, methods, uses and compounds to be employed inaccordance with the present invention may be retrieved from publiclibraries and databases, using for example electronic devices. Forexample, the public database “Medline”, available on the Internet, maybe utilized, for example underhttp://www.ncbi.nlm.nih.gov/PubMed/medline.html. Further databases andaddresses such as http://www.ncbi.nlm.nih.gov/ or listed at theEMBL-services homepage under http://www.embl.de/services/index.html areknown to the person skilled in the art and can also be obtained using,e.g., http://www.google.com.

The figures show:

FIG. 1

Fusion of the N-terminal amino acids 1-27 of primate CD3 epsilon to aheterologous soluble protein.

FIG. 2

The figure shows the average absorption values of quadruplicate samplesmeasured in an ELISA assay detecting the presence of a constructconsisting of the N-terminal amino acids 1-27 of the mature human CD3epsilon chain fused to the hinge and Fc gamma portion of human IgG1 anda C-terminal 6 Histidine tag in a supernatant of transiently transfected293 cells. The first column labeled “27 aa huCD3E” shows the averageabsorption value for the construct, the second column labeled “irrel.SN” shows the average value for a supernatant of 293 cells transfectedwith an irrelevant construct as negative control. The comparison of thevalues obtained for the construct with the values obtained for thenegative control clearly demonstrates the presence of the recombinantconstruct.

FIG. 3

The figure shows the average absorption values of quadruplicate samplesmeasured in an ELISA assay detecting the binding of the cross speciesspecific anti-CD3 binding molecules in form of crude preparations ofperiplasmatically expressed single-chain antibodies to a constructcomprising the N-terminal 1-27 amino acids of the mature human CD3epsilon chain fused to the hinge and Fc gamma portion of human IgG1 anda C-terminal His6 tag. The columns show from left to right the averageabsorption values for the specificities designated as A2J HLP, I2C HLPE2M HLP, F7O HLP, G4H HLP, H2C HLP, E1L HLP, F12Q HLP, F6A HLP and H1EHLP. The rightmost column labelled “neg. contr.” shows the averageabsorption value for the single-chain preparation of a murine anti-humanCD3 antibody as negative control. The comparison of the values obtainedfor the anti-CD3 specificities with the values obtained for the negativecontrol clearly demonstrates the strong binding of the anti-CD3specificities to the N-terminal 1-27 amino acids of the mature human CD3epsilon chain.

FIG. 4

Fusion of the N-terminal amino acids 1-27 of primate CD3 epsilon to aheterologous membrane bound protein.

FIG. 5

Histogram overlays of different transfectants tested in a FACS assaydetecting the presence of recombinant transmembrane fusion proteinsconsisting of cynomolgus EpCAM and the N-terminal 1-27 amino acids ofthe human, marmoset, tamarin, squirrel monkey and domestic swine CD3epsilon chain respectively. The histogram overlays from left to rightand top to bottom show the results for the transfectants expressing theconstructs comprising the human 27 mer, marmoset 27 mer, tamarin 27 mer,squirrel monkey 27 mer and swine 27 mer respectively. In the individualoverlays the thin line represents a sample incubated with PBS with 2%FCS instead of anti-Flag M2 antibody as negative control and the boldline shows a sample incubated with the anti-Flag M2 antibody. For eachconstruct the overlay of the histograms shows binding of the anti-FlagM2 antibody to the transfectants, which clearly demonstrates theexpression of the recombinant constructs on the transfectants.

FIG. 6

Histogram overlays of different transfectants tested in a FACS assaydetecting the binding of the cross-species specific anti-CD3 bindingmolecules in form of crude preparations of periplasmatically expressedsingle-chain antibodies to the N-terminal amino acids 1-27 of the human,marmoset, tamarin and squirrel monkey CD3 epsilon chain respectivelyfused to cynomolgus EpCAM.

FIG. 6A:

The histogram overlays from left to right and top to bottom show theresults for the transfectants expressing the 1-27 CD3-EpCAM comprisingthe human 27 mer tested with the CD3 specific binding moleculesdesignated H2C HLP, F12Q HLP, E2M HLP and G4H HLP respectively.

FIG. 6B:

The histogram overlays from left to right and top to bottom show theresults for the transfectants expressing the 1-27 CD3-EpCAM comprisingthe marmoset 27 mer tested with the CD3 specific binding moleculesdesignated H2C HLP, F12Q HLP, E2M HLP and G4H HLP respectively.

FIG. 6C:

The histogram overlays from left to right and top to bottom show theresults for the transfectants expressing the 1-27 CD3-EpCAM comprisingthe tamarin 27 mer tested with the CD3 specific binding moleculesdesignated H2C HLP, F12Q HLP, E2M HLP and G4H HLP respectively.

FIG. 6D:

The histogram overlays from left to right and top to bottom show theresults for the transfectants expressing the 1-27 CD3-EpCAM comprisingthe squirrel monkey 27 mer tested with the CD3 specific bindingmolecules designated H2C HLP, F12Q HLP, E2M HLP and G4H HLPrespectively.

FIG. 6E:

The histogram overlays from left to right and top to bottom show theresults for the transfectants expressing the 1-27 CD3-EpCAM comprisingthe swine 27 mer tested with the CD3 specific binding moleculesdesignated H2C HLP, F12Q HLP, E2M HLP and G4H HLP respectively.

In the individual overlays the thin line represents a sample incubatedwith a single-chain preparation of a murine anti-human CD3-antibody asnegative control and the bold line shows a sample incubated with therespective anti-CD3 binding molecules indicated. Considering the lack ofbinding to the swine 27 mer transfectants and the expression levels ofthe constructs shown in FIG. 5 the overlays of the histograms showspecific and strong binding of the tested anti-CD3 specificities of thefully cross-species specific human bispecific single chain antibodies tocells expressing the recombinant transmembrane fusion proteinscomprising the N-terminal amino acids 1-27 of the human, marmoset,tamarin and squirrel monkey CD3 epsilon chain respectively fused tocynomolgus EpCAM and show therefore multi primate cross-speciesspecificity of the anti-CD3 binding molecules.

FIG. 7

FACS assay for detection of human CD3 epsilon on transfected murine EL4T cells. Graphical analysis shows an overlay of histograms. The boldline shows transfected cells incubated with the anti-human CD3 antibodyUCHT-1. The thin line represents cells incubated with a mouse IgG1isotype control. Binding of the anti CD3 antibody UCHT1 clearly showsexpression of the human CD3 epsilon chain on the cell surface oftransfected murine EL4 T cells.

FIG. 8

Binding of cross-species specific anti CD3 antibodies to alanine-mutantsin an alanine scanning experiment. In the individual Figures the columnsshow from left to right the calculated binding values in arbitrary unitsin logarithmic scale for the wild-type transfectant (WT) and for allalanine-mutants from the position 1 to 27. The binding values arecalculated using the following formula:

${{value\_ Sample}\left( {x,y} \right)} = \frac{{{Sample}\left( {x,y} \right)} - {{neg\_ Contr}.(x)}}{\begin{matrix}{\left( {{U\; C\; H\; T} - {1(x)} - {{neg\_ Contr}.(x)}} \right)*} \\\frac{{{WT}(y)} - {{neg\_ Contr}.({wt})}}{{U\; C\; H\; T} - {1({wt})} - {{neg\_ Contr}.({wt})}}\end{matrix}}$

In this equation value_Sample means the value in arbitrary units ofbinding depicting the degree of binding of a specific anti-CD3 antibodyto a specific alanine-mutant as shown in the Figure, Sample means thegeometric mean fluorescence value obtained for a specific anti-CD3antibody assayed on a specific alanine-scanning transfectant, neg_Contr.means the geometric mean fluorescence value obtained for the negativecontrol assayed on a specific alanine-mutant, UCHT-1 means the geometricmean fluorescence value obtained for the UCHT-1 antibody assayed on aspecific alanine-mutant, WT means the geometric mean fluorescence valueobtained for a specific anti-CD3 antibody assayed on the wild-typetransfectant, x specifies the respective transfectant, y specifies therespective anti-CD3 antibody and wt specifies that the respectivetransfectant is the wild-type. Individual alanine-mutant positions arelabelled with the single letter code of the wild-type amino acid and thenumber of the position.

FIG. 8A:

The figure shows the results for cross-species specific anti CD3antibody A2J HLP expressed as chimeric IgG molecule. Reduced bindingactivity is observed for mutations to alanine at position 4(asparagine), at position 23 (threonine) and at position 25(isoleucine). Complete loss of binding is observed for mutations toalanine at position 1 (glutamine), at position 2 (aspartate), atposition 3 (glycine) and at position 5 (glutamate).

FIG. 8B:

The figure shows the results for cross-species specific anti CD3antibody E2M HLP, expressed as chimeric IgG molecule. Reduced bindingactivity is observed for mutations to alanine at position 4(asparagine), at position 23 (threonine) and at position 25(isoleucine). Complete loss of binding is observed for mutations toalanine at position 1 (glutamine), at position 2 (aspartate), atposition 3 (glycine) and at position 5 (glutamate).

FIG. 8C:

The figure shows the results for cross-species specific anti CD3antibody H2C HLP, expressed as chimeric IgG molecule. Reduced bindingactivity is observed for mutations to alanine at position 4(asparagine). Complete loss of binding is observed for mutations toalanine glutamine at position 1 (glutamine), at position 2 (aspartate),at position 3 (glycine) and at position 5 (glutamate).

FIG. 8D:

shows the results for cross-species specific anti CD3 antibody F12Q HLP,tested as periplasmatically expressed single-chain antibody. Completeloss of binding is observed for mutations to alanine at position 1(glutamine), at position 2 (aspartate), at position 3 (glycine) and atposition 5 (glutamate).

FIG. 9

FACS assay detecting the binding of the cross-species specific anti-CD3binding molecule H2C HLP to human CD3 with and without N-terminal His6tag.

Histogram overlays are performed of the EL4 cell line transfected withwild-type human CD3 epsilon chain (left histogram) or the human CD3epsilon chain with N-terminal His 6 tag (right histogram) tested in aFACS assay detecting the binding of cross-species specific bindingmolecule H2C HLP. Samples are incubated with an appropriate isotypecontrol as negative control (thin line), anti-human CD3 antibody UCHT-1as positive control (dotted line) and cross-species specific anti-CD3antibody H2C HLP in form of a chimeric IgG molecule (bold line).

Histogram overlays show comparable binding of the UCHT-1 antibody toboth transfectants as compared to the isotype control demonstratingexpression of both recombinant constructs. Histogram overlays also showbinding of the anti-CD3 binding molecule H2C HLP only to the wild-typehuman CD3 epsilon chain but not to the His6-human CD3 epsilon chain.These results demonstrate that a free N-terminus is essential forbinding of the cross-species specific anti-CD3 binding molecule H2C HLP.

FIG. 10

FACS binding analysis of designated cross-species specific bispecificsingle chain constructs to CHO cells transfected with the human MCSP D3,human CD3+ T cell line HPB-ALL, CHO cells transfected with cynomolgusMCSP D3 and a macaque T cell line 4119 LnPx. The FACS staining isperformed as described in Example 10. The thick line represents cellsincubated with 2 μg/ml purified protein that are subsequently incubatedwith the anti-his antibody and the PE labeled detection antibody. Thethin histogram line reflects the negative control: cells only incubatedwith the anti-his antibody and the detection antibody.

FIG. 11

FACS binding analysis of designated cross-species specific bispecificsingle chain constructs CHO cells transfected with the human MCSP D3,human CD3+ T cell line HPB-ALL, CHO cells transfected with cynomolgusMCSP D3 and a macaque T cell line 4119 LnPx. The FACS staining isperformed as described in Example 10. The thick line represents cellsincubated with 2 μg/ml purified protein that are subsequently incubatedwith the anti-his antibody and the PE labeled detection antibody. Thethin histogram line reflects the negative control: cells only incubatedwith the anti-his antibody and the detection antibody.

FIG. 12

FACS binding analysis of designated cross-species specific bispecificsingle chain constructs CHO cells transfected with the human MCSP D3,human CD3+ T cell line HPB-ALL, CHO cells transfected with cynomolgusMCSP D3 and a macaque T cell line 4119 LnPx. The FACS staining isperformed as described in Example 10. The thick line represents cellsincubated with 2 μg/ml purified monomeric protein that are subsequentlyincubated with the anti-his antibody and the PE labeled detectionantibody. The thin histogram line reflects the negative control: cellsonly incubated with the anti-his antibody and the detection antibody.

FIG. 13

Cytotoxicity activity induced by designated cross-species specific MCSPspecific single chain constructs redirected to indicated target celllines. A) Stimulated CD4-/CD56-human PBMCs are used as effector cells,CHO cells transfected with human MCSP D3 as target cells. B) The macaqueT cell line 4119 LnPx are used as effector cells, CHO cells transfectedwith cynomolgus MCSP D3 as target cells. The assay is performed asdescribed in Example 11.

FIG. 14

Cytotoxicity activity induced by designated cross-species specific MCSPspecific single chain constructs redirected to indicated target celllines. A) and B) The macaque T cell line 4119 LnPx are used as effectorcells, CHO cells transfected with cynomolgus MCSP D3 as target cells.The assay is performed as described in Example 11.

FIG. 15

Cytotoxicity activity induced by designated cross-species specific MCSPspecific single chain constructs redirected to indicated target celllines. A) and B) Stimulated CD4-/CD56-human PBMCs are used as effectorcells, CHO cells transfected with human MCSP D3 as target cells. Theassay is performed as described in Example 11.

FIG. 16

Cytotoxicity activity induced by designated cross-species specific MCSPspecific single chain constructs redirected to indicated target celllines. A) Stimulated CD4-/CD56-human PBMCs are used as effector cells,CHO cells transfected with human MCSP D3 as target cells. B) The macaqueT cell line 4119 LnPx are used as effector cells, CHO cells transfectedwith cynomolgus MCSP D3 as target cells. The assay is performed asdescribed in Example 11.

FIG. 17

Cytotoxicity activity induced by designated cross-species specific MCSPspecific single chain constructs redirected to indicated target celllines. A) Stimulated CD4-/CD56-human PBMCs are used as effector cells,CHO cells transfected with human MCSP D3 as target cells. B) The macaqueT cell line 4119 LnPx are used as effector cells, CHO cells transfectedwith cynomolgus MCSP D3 as target cells. The assay is performed asdescribed in Example 11.

FIG. 18

Plasma stability of MCSP and CD3 cross-species specific bispecificsingle chain antibodies tested by the measurement of cytotoxicityactivity induced by samples of the designated single chain constructsincubated with 50% human plasma at 37° C. and 4° C. for 24 hoursrespectively or with addition of 50% human plasma immediately prior tocytotoxicity testing or without addition of plasma. CHO cellstransfected with human MCSP are used as target cell line and stimulatedCD4-/CD56-human PBMCs are used as effector cells. The assay is performedas described in Example 12.

FIG. 19

Initial drop and recovery (i.e. redistribution) of absolute T cellcounts (open squares), in peripheral blood of B-NHL patients (patentnumbers 1, 7, 23, 30, 31, and 33 of Table 4), who had essentially nocirculating CD19-positive target B cells (filled triangles), during thestarting phase of intravenous infusion with the CD3 binding moleculeCD19×CD3 recognizing a conventional context dependent CD3 epitope.Absolute cell counts are given in 1000 cells per microliter blood. Thefirst data point shows baseline counts immediately prior to the start ofinfusion. The CD19×CD3 dose is given in parentheses beside the patientnumber.

FIG. 20

(A) Repeated T cell redistribution (open squares) in B-NHL patient #19(Table 4) who had no circulating CD19-positive target B cells (filledtriangles) and developed CNS symptoms under continuous intravenousinfusion with CD19×CD3 at a starting dose of 5 μg/m²/24 h for one dayfollowed by a sudden dose increase to 15 μg/m²/24 h. Absolute cellcounts are given in 1000 cells per microliter blood. The first datapoint shows baseline counts immediately prior to the start of infusion.After recovery of circulating T cells from the first episode ofredistribution triggered by the treatment start at 5 μg/m²/24 h thestepwise dose increase from 5 to 15 μg/m²/24 h triggered a secondepisode of T cell redistribution that was associated with thedevelopment of CNS symptoms dominated by confusion and disorientation.

(B) Repeated T cell redistribution in a B-NHL patient, who developed CNSsymptoms under repeated intravenous bolus infusion with CD19×CD3 at 1.5μg/m². Absolute cell counts are given in 1000 cells per microliterblood. The infusion time for each bolus administration was 2 to 4 hours.Vertical arrows indicate the start of bolus infusions. Data points atthe beginning of each bolus administration show the T cell countsimmediately prior to start of bolus infusion. Each bolus infusiontriggered an episode of T cell redistribution followed by recovery ofthe T cell counts prior to the next bolus infusion. Finally the thirdepisode of T cell redistribution was associated with the development ofCNS symptoms in this patient.

FIG. 21

Complex T cell redistribution pattern (open squares) in B-NHL patient#20 (Table 4) without circulating CD19-positive target B cells (filledtriangles), during ramp initiation of the CD19×CD3 infusion i.e. evengradual increase of flow-rate from almost zero to 15 μg/m²/24 h duringthe first 24 hours of treatment. Absolute cell counts are given in 1000cells per microliter blood. The first data point shows baseline countsimmediately prior to the start of infusion. The CD19×CD3 dose is givenin parentheses beside the patient number. T cells reappearing in thecirculating blood after the initial redistribution triggered by thefirst exposure to CD19×CD3 are partially induced to redisappear fromcirculating blood again by still increasing levels of CD19×CD3 duringthe ramp phase.

FIG. 22

T and B cell counts during treatment with CD19×CD3 of B-NHL patient #13(Table 4) who had a significant number of circulating CD19-positivetarget B (lymphoma) cells (filled triangles). Absolute cell counts aregiven in 1000 cells per microliter blood. The first data point showsbaseline counts immediately prior to the start of infusion. The CD19×CD3dose is given in parentheses beside the patient number. T cells (opensquares) disappear completely from the circulation upon start ofCD19×CD3 infusion and do not reappear until the circulatingCD19-positive B (lymphoma) cells (filled triangles) are depleted fromthe peripheral blood.

FIG. 23

Repeated T cell redistribution (open squares) in B-NHL patient #24(Table 4), who had essentially no circulating CD19-positive target Bcells (filled triangles) and developed CNS symptoms upon initiation ofCD19×CD3 infusion without additional HSA as required for stabilisationof the drug (upper panel). After first recovery of circulating T cellsfrom initial redistribution the uneven drug flow due to the lack ofstabilizing HSA triggered a second episode of T cell redistribution thatwas associated with the development of CNS symptoms dominated byconfusion and disorientation. When the same patient was restartedcorrectly with CD19×CD3 solution containing additional HSA for drugstabilisation, no repeated T cell redistribution was observed (lowerpanel) and the patient did not again develop any CNS symptoms. Absolutecell counts are given in 1000 cells per microliter blood. The first datapoint shows baseline counts immediately prior to the start of infusion.The CD19×CD3 dose is given in parentheses beside the patient number.

FIG. 24

Model of T cell adhesion to endothelial cells induced by monovalentbinding to context dependent CD3 epitopes. Monovalent interaction of aconventional CD3 binding molecule to its context dependent epitope onCD3 epsilon can lead to an allosteric change in the conformation of CD3followed by the recruitment of Nck2 to the cytoplasmic domain of CD3epsilon (Gil et al. (2002) Cell 109: 901). As Nck2 is directly linked tointegrins via PINCH and ILK (Legate et al. (2006) Nat Rev Mol Cell Biol7: 20), recruitment of Nck2 to the cytoplasmic domain of CD3 epsilonfollowing an allosteric change in the conformation of CD3 throughbinding of a conventional CD3 binding molecule (like the CD19×CD3 ofexample 13) to its context dependent epitope on CD3 epsilon, canincrease the adhesiveness of T cells to endothelial cells by transientlyswitching integrins on the T cell surface into their more adhesiveisoform via inside-out-signalling.

FIG. 25

Cytotoxic activity of CD33-AF5 VH-VL×I2C VH-VL test material used forthe in vivo study in cynomolgus monkeys as described in Example 14.Specific lysis of CD33-positive target cells was determined in astandard ⁵¹Chromium release assay at increasing concentrations ofCD33-AF5 VH-VL×I2C VH-VL. Assay duration was 18 hours. The macaque Tcell line 4119 LnPx was used as source of effector cells. CHO cellstransfected with cynomolgus CD33 served as target cells. Effector—totarget cell ratio (E:T-ratio) was 10:1. The concentration of CD33-AF5VH-VL×I2C VH-VL required for half-maximal target cell lysis (EC50) wascalculated from the dose response curve with a value of 2.7 ng/ml.

FIG. 26

(A) Dose- and time-dependent depletion of CD33-positive monocytes fromthe peripheral blood of cynomolgus monkeys through intravenouscontinuous infusion of CD33-AF5 VH-VL×I2C VH-VL as described in Example14. The percentage relative to baseline (i.e. 100%) of absolutecirculating CD33-positive monocyte counts after the duration oftreatment as indicated above the columns is shown for each of twocynomolgus monkeys per dose level. The dose level (i.e. infusionflow-rate) is indicated below the columns. No depletion of circulatingCD33-positive monocytes was observed in animals 1 and 2 treated for 7days at a dose of 30 μg/m²/24 h. In animals 3 and 4 treated for 7 daysat a dose of 60 μg/m²/24 h circulating CD33-positive monocyte countswere reduced to 68% and 40% of baseline, respectively. At 240 μg/m²/24 hcirculating CD33-positive monocytes were almost completely depleted fromthe peripheral blood after 3 days of treatment (animals 5 and 6). At1000 μg/m²/24 h depletion of circulating CD33-positive monocytes fromthe peripheral blood was completed already after 1 day of treatment(animals 7 and 8).

(B) Course of T cell and CD33-monocyte counts in peripheral blood of twocynomolgus monkeys during continuous infusion of CD33-AF5 VH-VL×I2CVH-VL for 14 days at 120 μg/m²/24 h. Absolute cell counts are given in1000 cells per microliter blood. The first data point shows baselinecounts immediately prior to the start of infusion. After initialmobilisation of CD33-monocytes during the first 12 hours upon start ofinfusion CD33-monocytes in peripheral blood (filled triangles) aredepleted by two thirds (animal 10) and 50% (animal 9) relative to therespective baseline counts during the further course of infusion.Circulating T cell counts (open squares) show a limited initial dropfollowed by recovery still during the presence of circulatingCD33-positive monocytic target cells.

FIG. 27

Cytotoxic activity of MCSP-G4 VH-VL×I2C VH-VL test material used for thein vivo study in cynomolgus monkeys as described in Example 15. Specificlysis of MCSP-positive target cells was determined in a standard⁵¹Chromium release assay at increasing concentrations of MCSP-G4VH-VL×I2C VH-VL. Assay duration was 18 hours. The macaque T cell line4119 LnPx was used as source of effector cells. CHO cells transfectedwith cynomolgus MCSP served as target cells. Effector- to target cellratio (E:T-ratio) was 10:1. The concentration of MCSP-G4 VH-VL×I2C VH-VLrequired for half-maximal target cell lysis (EC50) was calculated fromthe dose response curve with a value of 1.9 ng/ml.

FIG. 28

Absence of initial episodes of drop and subsequent recovery of absoluteT cell counts (i.e. redistribution) in peripheral blood of cynomolgusmonkeys during the starting phase of intravenous infusion with the CD3binding molecule MCSP-G4 VH-VL×I2C VH-VL recognizing an essentiallycontext independent CD3 epitope. Absolute cell counts are given in 1000cells per microliter blood. The first data point shows baseline countsimmediately prior to the start of infusion. The MCSP-G4 VH-VL×I2C VH-VLdose is given in parentheses beside the animal number. In the knownabsence of MCSP-positive target cells from the circulating blood ofcynomolgus monkeys there is no induction of T cell redistribution (i.e.an initial episode of drop and subsequent recovery of absolute T cellcounts) through target cell mediated crosslinking of CD3. Moreover,induction of T cell redistribution (i.e. an initial episode of drop andsubsequent recovery of absolute T cell counts) through a signal, whichthe T cells may receive through exclusive interaction with a CD3 bindingsite only, can be avoided by the use of CD3 binding molecules likeMCSP-G4 VH-VL×I2C VH-VL recognizing an essentially context independentCD3 epitope.

FIG. 29

FACS binding analysis of designated cross-species specific bispecificconstructs to CHO cells transfected with human CD33, the human CD3+ Tcell line HPB-ALL, CHO cells transfected with macaque CD33 and macaquePBMC respectively. The FACS staining is performed as described inExample 16.4. The bold lines represent cells incubated with 5 μg/mlpurified bispecific single chain construct or cell culture supernatantof transfected cells expressing the cross-species specific bispecificantibody constructs. The filled histograms reflect the negativecontrols. Supernatant of untransfected CHO cells was used as negativecontrol. For each cross-species specific bispecific single chainconstruct the overlay of the histograms shows specific binding of theconstruct to human and macaque CD33 and human and macaque CD3.

FIG. 30

The diagrams show results of chromium release assays measuring cytotoxicactivity induced by designated cross-species specific CD33 specificsingle chain constructs redirected to the indicated target cell lines.Effector cells were also used as indicated. The assays are performed asdescribed in Example 16.5. The diagrams clearly demonstrate for eachconstruct the potent recruitment of cytotoxic activity of human andmacaque effector cells against human and macaque CD33 transfected CHOcells, respectively.

FIG. 31

SDS PAGE gel and Western blot monitoring the purification of thecross-species specific bispecific single chain molecule designatedE292F3 HL×I2C HL. Samples from the eluate, the cell culture supernatant(SN) and the flow through of the column (FT) were analyzed as indicated.A protein marker (M) was applied as size reference. A strong proteinband with a molecular weight between 50 and 60 kDa in the SDS PAGE geldemonstrates the efficient purification of the cross-species specificbispecific single chain molecule to a very high degree of purity withthe one-step purification method described in Example 17.2. The Westernblot detecting the histidine₆ tag confirms the identity of the proteinband in the eluate as the cross-species specific bispecific single chainmolecule. The faint signal for the flow through sample in this sensitivedetection method further shows the nearly complete capture of bispecificsingle chain molecules by the purification method.

FIG. 32

SDS PAGE gel and Western blot monitoring the purification of thecross-species specific bispecific single chain molecule designatedV207C12 HL×H2C HL. Samples from the eluate, the cell culture supernatant(SN) and the flow through of the column (FT) were analyzed as indicated.A protein marker (M) was applied as size reference. A strong proteinband with a molecular weight between 50 and 60 kDa in the SDS PAGE geldemonstrates the efficient purification of the cross-species specificbispecific single chain molecule to a very high degree of purity withthe one-step purification method described in Example 17.2. The Westernblot detecting the histidine₆ tag confirms the identity of the proteinband in the eluate as the cross-species specific bispecific single chainmolecule. The faint signal for the flow through sample in this sensitivedetection method further shows the nearly complete capture of bispecificsingle chain molecules by the purification method.

FIG. 33

SDS PAGE gel and Western blot monitoring the purification of thecross-species specific bispecific single chain molecule designatedAF5HL×F12QHL. Samples from the eluate, the cell culture supernatant (SN)and the flow through of the column (FT) were analyzed as indicated. Aprotein marker (M) was applied as size reference. A strong protein bandwith a molecular weight between 50 and 60 kDa in the SDS PAGE geldemonstrates the efficient purification of the cross-species specificbispecific single chain molecule to a very high degree of purity withthe one-step purification method described in Example 17.2. The Westernblot detecting the histidine₆ tag confirms the identity of the proteinband in the eluate as the cross-species specific bispecific single chainmolecule. The signal in the flow through sample in this sensitivedetection method is explained by saturation of the affinity column dueto the high concentration of bispecific single chain molecules in thesupernatant.

FIG. 34

Standard curve of AF5HL×I2CHL in 50% macaque monkey serum. The upperdiagram shows the standard curve generated for the assay as described inExample 18.2.

The lower diagram shows results for quality control samples ofAF5HL×I2CHL in 50% macaque monkey serum. The recovery rates are above90% for the high and mid QC sample and above 80% for the low QC sample.

Thus the assay allows for detection of AF5HL×I2CHL in serum samples inthe range from 10 ng/ml to 200 ng/ml (before dilution).

FIG. 35

Standard curve of MCSP-G4 HL×I2C HL in 50% macaque monkey serum. Theupper diagram shows the standard curve generated for the assay asdescribed in Example 18.2.

The lower diagram shows results for quality control samples of MCSP-G4HL×I2C HL in 50% macaque monkey serum. The recovery rates are above 98%for the high and mid QC sample and above 85% for the low QC sample.

Thus the assay allows for detection of MCSP-G4 HL×I2C HL in serumsamples in the range from 10 ng/ml to 200 ng/ml (before dilution).

FIG. 36

FACS binding analysis of an anti-Flag antibody to CHO cells transfectedwith the 1-27 N-terminal amino acids of CD3 epsilon of the designatedspecies fused to cynomolgus EpCAM. The FACS staining was performed asdescribed in Example 19.1. The bold lines represent cells incubated withthe anti-Flag antibody. The filled histograms reflect the negativecontrols. PBS with 2% FCS was used as negative control. The histogramsshow strong and comparable binding of the anti-Flag antibody to alltransfectants indicating strong and equal expression of the transfectedconstructs.

FIG. 37

FACS binding analysis of the I2C IgG1 construct to CHO cells expressingthe 1-27 N-terminal amino acids of CD3 epsilon of the designated speciesfused to cynomolgus EpCAM. The FACS staining is performed as describedin Example 19.3. The bold lines represent cells incubated with 50 μlcell culture supernatant of cells expressing the I2C IgG1 construct. Thefilled histograms reflect the negative control. Cells expressing the1-27 N-terminal amino acids of CD3 epsilon of swine fused to cynomolgusEpCAM were used as negative control. In comparison with the negativecontrol the histograms clearly demonstrate binding of the I2C IgG1construct to 1-27 N-terminal amino acids of CD3 epsilon of human,marmoset, tamarin and squirrel monkey.

FIG. 38

FACS binding analysis of the I2C IgG1 construct as described in Example19.2 to human CD3 with and without N-terminal His6 tag as described inExamples 6.1 and 5.1 respectively. The bold lines represent cellsincubated with the anti-human CD3 antibody UCHT-1, the penta-Hisantibody (Qiagen) and cell culture supernatant of cells expressing theI2C IgG1 construct respectively as indicated. The filled histogramsreflect cells incubated with an irrelevant murine IgG1 antibody asnegative control.

The upper two histogram overlays show comparable binding of the UCHT-1antibody to both transfectants as compared to the isotype controldemonstrating expression of both recombinant constructs. The centrehistogram overlays show binding of the penta his antibody to the cellsexpressing the His6-human CD3 epsilon chain (His6-CD3) but not to thecells expressing the wild-type CD3 epsilon chain (WT-CD3). The lowerHistogram overlays show binding of the I2C IgG1 construct to thewild-type human CD3 epsilon chain but not to the His6-human CD3 epsilonchain. These results demonstrate that a free N-terminus is essential forbinding of the cross-species specific anti-CD3 binding molecule I2C tothe CD3 epsilon chain.

FIG. 39

FACS binding analysis of designated cross-species specific bispecificsingle chain constructs to CHO cells transfected with human MCSP D3, thehuman CD3+ T cell line HPB-ALL, CHO cells transfected with macaque MCSPD3 and the macaque T cell line 4119 LnPx respectively. The FACS stainingwas performed as described in Example 10. The bold lines representscells incubated with 2 μg/ml purified bispecific single chain constructor cell supernatant containing the bispecific single chain constructrespectively. The filled histograms reflect the negative controls.Supernatant of untransfected CHO cells was used as negative control forbinding to the T cell lines. A single chain construct with irrelevanttarget specificity was used as negative control for binding to the MCSPD3 transfected CHO cells. For each cross-species specific bispecificsingle chain construct the overlay of the histograms shows specificbinding of the construct to human and macaque MCSP D3 and human andmacaque CD3.

FIG. 40

Cytotoxic activity induced by designated cross-species specific MCSP D3specific single chain constructs redirected to the indicated target celllines. Effector cells and effector to target ratio were also used asindicated. The assay is performed as described in Example 11. Thediagrams clearly demonstrate potent cross-species specific recruitmentof cytotoxic activity by each construct.

FIG. 41

FACS binding analysis of designated cross-species specific bispecificsingle chain constructs to CHO cells transfected with human CD33, thehuman CD3+ T cell line HPB-ALL, CHO cells transfected with macaque CD33and macaque PBMC respectively. The FACS staining was performed asdescribed in Example 21.2. The bold lines represent cells incubated withcell culture supernatant of transfected cells expressing thecross-species specific bispecific antibody constructs. The filledhistograms reflect the negative controls. Supernatant of untransfectedCHO cells was used as negative control. For each cross-species specificbispecific single chain construct the overlay of the histograms showsspecific binding of the construct to human and macaque CD33 and humanand macaque CD3.

FIG. 42

The diagrams show results of chromium release assays measuring cytotoxicactivity induced by designated cross-species specific CD33 specificsingle chain constructs redirected to the indicated target cell lines.Effector cells were also used as indicated. The assays are performed asdescribed in Example 21.3. The diagrams clearly demonstrate for eachconstruct the potent recruitment of cytotoxic activity of human andmacaque effector cells against human and macaque CD33 transfected CHOcells, respectively.

FIG. 43

T cell redistribution in a chimpanzee under weekly intravenous bolusinfusion with PBS/5% HSA and PBS/5% HSA plus single-chainEpCAM/CD3-bispecific antibody construct at doses of 1.6, 2.0, 3.0 and4.5 μg/kg. The infusion time for each bolus administration was 2 hours.Vertical arrows indicate the start of bolus infusions. Data points atthe beginning of each bolus administration show the T cell countsimmediately prior to start of bolus infusion. Each bolus infusion of thesingle-chain EpCAM/CD3-bispecific antibody construct, which recognizes aconventional context dependent CD3 epitope, triggered an episode of Tcell redistribution followed by recovery of T cells to baseline valuesprior to the next bolus infusion.

FIG. 44

CD3 specific ELISA analysis of periplasmic preparations containing Flagtagged scFv protein fragments from selected clones. Periplasmicpreparations of soluble scFv protein fragments were added to wells of anELISA plate, which had been coated with soluble human CD3 epsilon (aa1-27)-Fc fusion protein and had been additionally blocked with PBS 3%BSA. Detection was performed by a monoclonal anti Flag-Biotin-labeledantibody followed by peroxidase-conjugated Streptavidin. The ELISA wasdeveloped by an ABTS substrate solution. The OD values (y axis) weremeasured at 405 nm by an ELISA reader. Clone names are presented on thex axis.

FIG. 45

ELISA analysis of periplasmic preparations containing Flag tagged scFvprotein fragments from selected clones. The same periplasmicpreparations of soluble scFv protein fragments as in FIG. 44 were addedto wells of an ELISA plate which had not been coated with human CD3epsilon (aa 1-27)-Fc fusion protein but with huIgG1 (Sigma) and blockedwith 3% BSA in PBS.

Detection was performed by a monoclonal anti Flag-Biotin-labeledantibody followed by peroxidase-conjugated Streptavidin. The ELISA wasdeveloped by an ABTS substrate solution. The OD values (y axis) weremeasured at 405 nm by an ELISA reader. Clone names are presented on thex axis.

FIG. 46/1

FACS binding analysis of designated cross-species specific single domainbispecific single chain constructs to CHO cells transfected with humanEGFR, human CD3+ T cell line HPB-ALL, CHO cells transfected with macaqueEGFR and a macaque T cell line 4119 LnPx. The FACS staining wasperformed as described in Example 24.5. The thick line represents cellsincubated with cell culture supernatant containing the construct to betested that were subsequently incubated with the anti-His antibody andthe PE labeled detection antibody. The thin histogram line reflects thenegative control: cells only incubated with the anti-his antibody andthe detection antibody.

FIG. 46/2

FACS binding analysis of designated cross-species specific single domainbispecific single chain constructs to CHO cells transfected with thehuman EGFR, human CD3+ T cell line HPB-ALL, CHO cells transfected withmacaque EGFR and a macaque T cell line 4119 LnPx. The FACS staining wasperformed as described in Example 24. 5. The thick line represents cellsincubated with cell culture supernatant containing the construct to betested that were subsequently incubated with the anti-His antibody andthe PE labeled detection antibody. The thin histogram line reflects thenegative control: cells only incubated with the anti-his antibody andthe detection antibody.

FIG. 47/1

Cytotoxic activity induced by designated cross-species specific singledomain bispecific single chain constructs redirected to indicated targetcell lines. A) Stimulated CD4-/CD56-human PBMCs were used as effectorcells, CHO cells transfected with human EGFR as target cells. B) Themacaque T cell line 4119 LnPx was used as source of effector cells, CHOcells transfected with macaque EGFR were used as target cells. The assaywas performed as described in Example 24.6.

FIG. 47/2

Cytotoxic activity induced by designated cross-species specific singledomain bispecific single chain constructs redirected to indicated targetcell lines. A) Stimulated CD4-/CD56-human PBMCs were used as effectorcells, CHO cells transfected with human EGFR as target cells. B) Themacaque T cell line 4119 LnPx was used as source of effector cells, CHOcells transfected with macaque EGFR as target cells. The assay wasperformed as described in Example 24.6.

FIG. 48

52 days after the first immunization antibody serum titers against theCD3-positive human T cell line HPBaII and the macaque CD3-positive Tcell line 4119LnPx were tested in flow cytometry according to standardprotocols. To this end 200.000 cells of the respective cell lines wereincubated for 30 min on ice with 50 μl of serum of the immunized animalsdiluted 1:1000 in PBS with 2% FCS. The cells were washed twice in PBSwith 2% FCS and binding of serum antibodies was detected with a FITCconjugated Goat anti-Llama IgG-H&L Antibody diluted 1:100 in 50 μl PBSwith 2% FCS. Serum of the animals obtained prior to immunization wasused as a negative control (filled curve). Flow cytometry was performedand analyzed. Reactivity to the CD3-positive human T cell line HPBaIIand the CD3-positive macaque T cell line 4119LnPx of a serum sample ofone exemplary animal obtained 52 days after the first immunization wasclearly detectable (bold lines).

FIG. 49

Binding of crude preparations of periplasmatically expressed anti-CD3single domain antibody CD3 3D-H11 to human and non-chimpanzee primateCD3 was tested by flowcytometry on the CD3 positive human T cellleukemia cell line HPB-ALL and the CD3 positive macaque T cell line4119LnPx. For flow cytometry 2.5×10⁵ cells were incubated with 50 ulperiplasmic supernatant. The binding of the constructs was detected withan anti-His antibody followed by PE conjugated goat anti-mouse IgG. Thesamples were measured on a FACSscan. The filled curves represent thenegative controls; the thick lines represent CD3 3D-H11. The overlays ofthe histograms show cross-species specific binding of anti-CD3 singledomain binder CD3 3D-H11 to human and macaque T cells and no binding toCD3 negative CHO cells.

FIG. 50

Binding of crude preparations of periplasmatically expressed anti-CD3single domain antibody CD3 3D-H11 to immobilized human 1-27 CD3-Fcfusion protein and human 1-27 CD3 BSA conjugate was tested in an ELISAassay. Antigen was immobilized over night and the subsequently blockedwells were then were incubated with crude preparations ofperiplasmatically expressed single domain antibody at room temperature.After washing with PBS/Tween wells were incubated with peroxidaseconjugated anti-Flag M2 antibody and after washing incubated withl ofthe SIGMAFAST OPD (OPD [o-Phenylenediamine dihydrochloride] substratesolution (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) according tothe manufacturers protocol. Color reaction was stopped and the platemeasured on a PowerWaveX microplate spectrophotometer at 490 nm andsubtraction of background absorption at 620 nm. Binding of detectionantibody alone was substracted. Binding of anti-CD3 single domainantibody CD3 3D-H11 to 1-27 CD3-Fc fusion protein as well as to 1-27 CD3BSA conjugate could clearly be detected, whereas the signal on theblocking agent alone was negligible.

The present invention is additionally described by way of the followingillustrative non-limiting examples that provide a better understandingof the present invention and of its many advantages.

EXAMPLES 1. Identification of CD3Epsilon Sequences from Blood Samples ofNon-Human Primates

Blood samples of the following non-human primates were used forCD3epsilon-identification: Callithrix jacchus, Saguinus oedipus andSaimiris ciureus. Fresh heparin-treated whole blood samples wereprepared for isolating total cellular RNA according to manufacturer'sprotocol (QIAamp RNA Blood Mini Kit, Qiagen). The extracted mRNA wastranscribed into cDNA according to published protocols. In brief, 10 μlof precipitated RNA was incubated with 1.2 μl of 10× hexanucleotide mix(Roche) at 70° C. for 10 minutes and stored on ice. A reaction mixconsisting of 4 μl of 5× superscript II buffer, 0.2 μl of 0.1Mdithiothreitole, 0.8 μl of superscript II (Invitrogen), 1.2 μl ofdesoxyribonucleoside triphosphates (25 μM), 0.8 μl of RNase Inhibitor(Roche) and 1.8 μl of DNase and RNase free water (Roth) was added. Thereaction mix was incubated at room temperature for 10 minutes followedby incubation at 42° C. for 50 minutes and at 90° C. for 5 minutes. Thereaction was cooled on ice before adding 0.8 μl of RNaseH (1 U/μl,Roche) and incubated for 20 minutes at 37° C.

The first-strand cDNAs from each species were subjected to separate35-cycle polymerase chain reactions using Taq DNA polymerase (Sigma) andthe following primer combination designed on database research: forwardprimer 5′-AGAGTTCTGGGCCTCTGC-3′ (SEQ ID NO: 253); reverse primer5′-CGGATGGGCTCATAGTCTG-3′ (SEQ ID NO: 254). The amplified 550 bp-bandswere gel purified (Gel Extraction Kit, Qiagen) and sequenced(Sequiserve, Vaterstetten/Germany, see sequence listing).

CD3epsilon Callithrix jacchus NucleotidesCAGGACGGTAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACATGAAATAAAATGGCTCGTAAATAGTCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAGGACTTTTCGGAAATGGAGCAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACTCCCGCAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGTGAGAACTGCGTGGAGGTG GAT Amino acids (SEQ IDNO: 3) QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPAEEASHYLYLKARVCENCVEVD CD3epsilon Saguinusoedipus Nucleotides CAGGACGGTAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACATGAAATAAAATGGCTTGTAAATAGTCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAGGATTTTTCGGAAATGGAGCAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACTCCCGCAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGTGAGAACTGCGTGGAG GTGGAT Amino acids (SEQID NO: 5) QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPAEEASHYLYLKARVCENCVEVD CD3epsilon Saimirisciureus Nucleotides CAGGACGGTAATGAAGAGATTGGTGATACTACCCAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACATGCCCTCGGTATGATGGACAGGAAATAAAATGGCTCGTAAATGATCAAAACAAAGAAGGTCATGAGGACCACCTGTTACTGGAAGATTTTTCAGAAATGGAACAAAGTGGTTATTATGCCTGCCTCTCCAAAGAGACCCCCACAGAAGAGGCGAGCCATTATCTCTACCTGAAGGCAAGAGTGTGTGAGAACTGCGTGGAGG TGGAT Amino acids (SEQ IDNO: 7) QDGNEEIGDTTQNPYKVSISGTTVTLTCPRYDGQEIKWLVNDQNKEGHEDHLLLEDFSEMEQSGYYACLSKETPTEEASHYLYLKARVCENCVEVD

2. Generation of Cross-Species Specific Single Chain Antibody Fragments(scFv) Binding to the N-Terminal Amino Acids 1-27 of CD3Epsilon of Manand Different Non-Chimpanzee Primates

2.1. Immunization of Mice Using the N-Terminus of CD3Epsilon Separatedfrom its Native CD3-Context by Fusion to a Heterologous Soluble Protein

Ten weeks old F1 mice from balb/c×C57black crossings were immunized withthe CD3epsilon-Fc fusion protein carrying themost N-terminal amino acids1-27 of the mature CD3epsilon chain (1-27 CD3-Fc) of man and/or saimirisciureus. To this end 40 μg of the 1-27 CD3-Fc fusion protein with 10nmol of a thioate-modified CpG-Oligonucleotide(5′-tccatgacgttcctgatgct-3′) (SEQ ID No. 343) in 300 ul PBS wereinjected per mouse intra-peritoneally. Mice receive boosterimmunizations after 21, 42 and optionally 63 days in the same way. Tendays after the first booster immunization, blood samples were taken andantibody serum titer against 1-27 CD3-Fc fusion protein iwa tested byELISA. Additionally, the titer against the CD3-positive human T cellline HPBall was tested in flow cytometry according to standardprotocols. Serum titers were significantly higher in immunized than innon-immunized animals.

2.2. Generation of an Immune Murine Antibody scFv Library: Constructionof a Combinatorial Antibody Library and Phage Display

Three days after the last injection the murine spleen cells wereharvested for the preparation of total RNA according to standardprotocols.

A library of murine immunoglobuline (Ig) light chain (kappa) variableregion (VK) and Ig heavy chain variable region (VH) DNA-fragments wasconstructed by RT-PCR on murine spleen RNA using VK- and VH specificprimer. cDNA was synthesized according to standard protocols.

The primers were designed in a way to give rise to a 5′-XhoI and a3′-BstEII recognition site for the amplified heavy chain V-fragments andto a 5′-SacI and a 3′-SpeI recognition site for amplified VK DNAfragments.

For the PCR-amplification of the VH DNA-fragments eight different5′-VH-family specific primers (MVH1(GC)AG GTG CAG CTC GAG GAG TCA GGACCT (SEQ ID No. 344); MVH2 GAG GTC CAG CTC GAG CAG TCT GGA CCT (SEQ IDNo. 345); MVH3 CAG GTC CAA CTC GAG CAG CCT GGG GCT (SEQ ID No. 346);MVH4 GAG GTT CAG CTC GAG CAG TCT GGG GCA (SEQ ID No. 347); MVH5 GA(AG)GTG AAG CTC GAG GAG TCT GGA GGA (SEQ ID No. 348); MVH6 GAG GTG AAG CTTCTC GAG TCT GGA GGT (SEQ ID No. 349); MVH7 GAA GTG AAG CTC GAG GAG TCTGGG GGA (SEQ ID No. 350); MVH8 GAG GTT CAG CTC GAG CAG TCT GGA GCT (SEQID No. 351)) were each combined with one 3′-VH primer (3′MuVHBstEII tgagga gac ggt gac cgt ggt ccc ttg gcc cca g (SEQ ID No. 352)); for the PCRamplification of the VK-chain fragments seven different 5′-VK-familyspecific primers (MUVK1 CCA GTT CCG AGC TCG TTG TGA CTC AGG AAT CT (SEQID No. 353); MUVK2 CCA GTT CCG AGC TCG TGT TGA CGC AGC CGC CC (SEQ IDNo. 354); MUVK3 CCA GTT CCG AGC TCG TGC TCA CCC AGT CTC CA (SEQ ID No.355); MUVK4 CCA GTT CCG AGC TCC AGA TGA CCC AGT CTC CA (SEQ ID No. 356);MUVK5 CCA GAT GTG AGC TCG TGA TGA CCC AGA CTC CA (SEQ ID No. 357); MUVK6CCA GAT GTG AGC TCG TCA TGA CCC AGT CTC CA (SEQ ID No. 358); MUVK7 CCAGTT CCG AGC TCG TGA TGA CAC AGT CTC CA (SEQ ID No. 359)) were eachcombined with one 3′-VK primer (3′MuVkHindIII/BsiW1 tgg tgc act agt cgtacg ttt gat ctc aag ctt ggt ccc (SEQ ID No. 360)).

The following PCR program was used for amplification: denaturation at94° C. for 20 sec; primer annealing at 52° C. for 50 sec and primerextension at 72° C. for 60 sec and 40 cycles, followed by a 10 min finalextension at 72° C.

450 ng of the kappa light chain fragments (SacI-SpeI digested) wereligated with 1400 ng of the phagemid pComb3H5Bhis (SacI-SpeI digested;large fragment). The resulting combinatorial antibody library was thentransformed into 300 ul of electrocompetent Escherichia coli XL1 Bluecells by electroporation (2.5 kV, 0.2 cm gap cuvette, 25 uFD, 200 Ohm,Biorad gene-pulser) resulting in a library size of more than 10⁷independent clones. After one hour of phenotype expression, positivetransformants were selected for carbenicilline resistance encoded by thepComb3H5BHis vector in 100 ml of liquid super broth (SB)-culture overnight. Cells were then harvested by centrifugation and plasmidpreparation was carried out using a commercially available plasmidpreparation kit (Qiagen).

2800 ng of this plasmid-DNA containing the VK-library (XhoI-BstEIIdigested; large fragment) were ligated with 900 ng of the heavy chainV-fragments (XhoI-BstEII digested) and again transformed into two 300 ulaliquots of electrocompetent E. coli XL1 Blue cells by electroporation(2.5 kV, 0.2 cm gap cuvette, 25 uFD, 200 Ohm) resulting in a total VH-VKscFv (single chain variable fragment) library size of more than 10⁷independent clones.

After phenotype expression and slow adaptation to carbenicillin, the E.coli cells containing the antibody library were transferred intoSB-Carbenicillin (50 ug/mL) selection medium. The E. coli cellscontaining the antibody library wass then infected with an infectiousdose of 10¹² particles of helper phage VCSM13 resulting in theproduction and secretion of filamentous M13 phage, wherein phageparticle contains single stranded pComb3H5BHis-DNA encoding a murinescFv-fragment and displayed the corresponding scFv-protein as atranslational fusion to phage coat protein III. This pool of phagesdisplaying the antibody library was later used for the selection ofantigen binding entities.

2.3. Phage Display Based Selection of CD3-Specific Binders

The phage library carrying the cloned scFv-repertoire was harvested fromthe respective culture supernatant by PEG8000/NaCl precipitation andcentrifugation. Approximately 10¹¹ to 10¹² scFv phage particles wereresuspended in 0.4 ml of PBS/0.1% BSA and incubated with 10⁵ to 10⁷Jurkat cells (a CD3-positive human T-cell line) for 1 hour on ice underslow agitation. These Jurkat cells were grown beforehand in RPMI mediumenriched with fetal calf serum (10%), glutamine andpenicillin/streptomycin, harvested by centrifugation, washed in PBS andresuspended in PBS/1% FCS (containing Na Azide). scFv phage which do notspecifically bind to the Jurkat cells were eliminated by up to fivewashing steps with PBS/1% FCS (containing Na Azide). After washing,binding entities were eluted from the cells by resuspending the cells inHCl-glycine pH 2.2 (10 min incubation with subsequent vortexing) andafter neutralization with 2 M Tris pH 12, the eluate was used forinfection of a fresh uninfected E. coli XL1 Blue culture (OD600>0.5).The E. coli culture containing E. coli cells successfully transducedwith a phagemid copy, encoding a human scFv-fragment, were againselected for carbenicillin resistance and subsequently infected withVCMS13 helper phage to start the second round of antibody display and invitro selection. A total of 4 to 5 rounds of selections were carriedout, normally.

2.4. Screening for CD3-Specific Binders

Plasmid DNA corresponding to 4 and 5 rounds of panning was isolated fromE. coli cultures after selection. For the production of solublescFv-protein, VH-VL-DNA fragments were excised from the plasmids(XhoI-SpeI). These fragments were cloned via the same restriction sitesin the plasmid pComb3H5BFlag/His differing from the originalpComb3H5BHis in that the expression construct (e.g. scFv) includes aFlag-tag (TGD YKDDDDK) between the scFv and the His6-tag and theadditional phage proteins were deleted. After ligation, each pool(different rounds of panning) of plasmid DNA was transformed into 100 μlheat shock competent E. coli TG1 or XLI blue and plated ontocarbenicillin LB-agar. Single colonies were picked into 100 ul of LBcarb (50 ug/ml).

E. coli transformed with pComb3H5BHis containing a VL- and VH-segmentproduce soluble scFv in sufficient amounts after excision of the geneIII fragment and induction with 1 mM IPTG. Due to a suitable signalsequence, the scFv-chain was exported into the periplasma where it foldsinto a functional conformation.

Single E. coli TG1 bacterial colonies from the transformation plateswere picked for periplasmic small scale preparations and grown inSB-medium (e.g. 10 ml) supplemented with 20 mM MgCl2 and carbenicillin50 μg/ml (and re-dissolved in PBS (e.g. 1 ml) after harvesting. By fourrounds of freezing at −70° C. and thawing at 37° C., the outer membraneof the bacteria was destroyed by temperature shock and the solubleperiplasmic proteins including the scFvs were released into thesupernatant. After elimination of intact cells and cell-debris bycentrifugation, the supernatant containing the human anti-humanCD3-scFvs was collected and used for further examination.

2.5. Identification of CD3-Specific Binders

Binding of the isolated scFvs was tested by flow cytometry on eukaryoticcells, which on their surface express a heterologous protein displayingat its N-terminus the first 27 N-terminal amino acids of CD3epsilon.

As described in Example 4, the first amino acids 1-27 of the N-terminalsequence of the mature CD3 epsilon chain of the human T cell receptorcomplex (amino acid sequence: QDGNEEMGGITQTPYKVSISGTTVILT SEQ ID NO: 2)were fused to the N-terminus of the transmembrane protein EpCAM so thatthe N-terminus was located at the outer cell surface. Additionally, aFLAG epitope was inserted between the N-terminal 1-27 CD3epsilonsequence and the EpCAM sequence. This fusion product was expressed inhuman embryonic kidney (HEK) and chinese hamster ovary (CHO) cells.

Eukaryotic cells displaying the 27 most N-terminal amino acids of matureCD3epsilon of other primate species were prepared in the same way forSaimiri ciureus (Squirrel monkey) (CD3 epsilon N-terminal amino acidsequence: QDGNEEIGDTTQNPYKVSISGTTVTLT SEQ ID NO: 8), for Callithrixjacchus (CD3 epsilon N-terminal amino acid sequence:QDGNEEMGDTTQNPYKVSISGTTVTLT SEQ ID NO: 4) and for Saguinus oedipus (CD3epsilon N-terminal amino acid sequence: QDGNEEMGDTTQNPYKVSISGTTVTLT SEQID NO: 6).

For flow cytometry 2.5×10⁵ cells are incubated with 50 ul supernatant orwith 5 μg/ml of the purified constructs in 50 μl PBS with 2% FCS. Thebinding of the constructs was detected with an anti-His antibody(Penta-His Antibody, BSA free, Qiagen GmbH, Hilden, FRG) at 2 μg/ml in50 μl PBS with 2% FCS. As a second step reagent aR-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment, goatanti-mouse IgG (Fc-gamma fragment specific), diluted 1:100 in 50 μl PBSwith 2% FCS (Dianova, Hamburg, FRG) was used. The samples were measuredon a FACSscan (BD biosciences, Heidelberg, FRG).

Binding was always confirmed by flowcytometry as described in theforegoing paragraph on primary T cells of man and different primates(e.g. saimiris ciureus, callithrix jacchus, saguinus oedipus).

2.6. Generation of Human/Humanized Equivalents of Non-Human CD3EpsilonSpecific scFvs

The VH region of the murine anti-CD3 scFv was aligned against humanantibody germline amino acid sequences. The human antibody germline VHsequence was chosen which has the closest homology to the non-human VHand a direct alignment of the two amino acid sequences was performed.There were a number of framework residues of the non-human VH thatdiffer from the human VH framework regions (“different frameworkpositions”). Some of these residues may contribute to the binding andactivity of the antibody to its target.

To construct a library that contain the murine CDRs and at everyframework position that differs from the chosen human VH sequence bothpossibilities (the human and the maternal murine amino acid residue),degenerated oligonucleotides were synthesized. These oligonucleotidesincorporate at the differing positions the human residue with aprobability of 75% and the murine residue with a probability of 25%. Forone human VH e.g. six of these oligonucleotides had to be synthesizedthat overlap in a terminal stretch of approximately 20 nucleotides. Tothis end every second primer was an antisense primer. Restriction sitesneeded for later cloning within the oligonucleotides were deleted.

These primers may have a length of 60 to 90 nucleotides, depending onthe number of primers that were needed to span over the whole Vsequence.

These e.g. six primers were mixed in equal amounts (e.g. 1 μl of eachprimer (primer stocks 20 to 100 μM) to a 20 μl PCR reaction) and addedto a PCR mix consisting of PCR buffer, nucleotides and Taq polymerase.This mix was incubated at 94° C. for 3 minutes, 65° C. for 1 minute, 62°C. for 1 minute, 59° C. for 1 minute, 56° C. for 1 minute, 52° C. for 1minute, 50° C. for 1 minute and at 72° C. for 10 minutes in a PCRcycler. Subsequently the product was run in an agarose gelelectrophoresis and the product of a size from 200 to 400 isolated fromthe gel according to standard methods.

This PCR product was then used as a template for a standard PCR reactionusing primers that incorporate N-terminal and C-terminal suitablecloning restriction sites. The DNA fragment of the correct size (for aVH approximately 350 nucleotides) was isolated by agarose gelelectrophoresis according to standard methods. In this way sufficient VHDNA fragment was amplified. This VH fragment was now a pool of VHfragments that have each one a different amount of human and murineresidues at the respective differing framework positions (pool ofhumanized VH). The same procedure was performed for the VL region of themurine anti-CD3 scFv (pool of humanized VL).

The pool of humanized VH was then combined with the pool of humanized VLin the phage display vector pComb3H5Bhis to form a library of functionalscFvs from which—after display on filamentous phage—anti-CD3 binderswere selected, screened, identified and confirmed as described above forthe parental non-human (murine) anti-CD3 scFv. Single clones were thenanalyzed for favorable properties and amino acid sequence. Those scFvswhich were closest in amino acid sequence homology to human germlineV-segments are preferred particularly those wherein at least one CDRamong CDR I and II of VH and CDR I and II of VLkappa or CDR I and II ofVLlambda shows more than 80% amino acid sequence identity to the closestrespective CDR of all human germline V-segments. Anti-CD3 scFvs wereconverted into recombinant bispecific single chain antibodies asdescribed in the following Examples 9, 16, and 24.

3. Generation of a Recombinant Fusion Protein of the N-Terminal AminoAcids 1-27 of the Human CD3 Epsilon Chain Fused to the Fc-Part of anIgG1 (1-27 CD3-Fc) 3.1. Cloning and Expression of 1-27 CD3-Fc

The coding sequence of the 1-27 N-terminal amino acids of the human CD3epsilon chain fused to the hinge and Fc gamma region of humanimmunoglobulin IgG1 as well as an 6 Histidine Tag were obtained by genesynthesis according to standard protocols (cDNA sequence and amino acidsequence of the recombinant fusion protein are listed under SEQ ID NOs230 and 229). The gene synthesis fragment was designed as to containfirst a Kozak site for eukaryotic expression of the construct, followedby an 19 amino acid immunoglobulin leader peptide, followed in frame bythe coding sequence of the first 27 amino acids of the extracellularportion of the mature human CD3 epsilon chain, followed in frame by thecoding sequence of the hinge region and Fc gamma portion of human IgG1,followed in frame by the coding sequence of a 6 Histidine tag and a stopcodon (FIG. 1). The gene synthesis fragment was also designed as tointroduce restriction sites at the beginning and at the end of the cDNAcoding for the fusion protein. The introduced restriction sites, EcoRIat the 5′ end and SalI at the 3′ end, are utilized in the followingcloning procedures. The gene synthesis fragment was cloned via EcoRI andSalI into a plasmid designated pEF-DHFR (pEF-DHFR is described in Macket al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) followingstandard protocols. A sequence verified plasmid was used fortransfection in the FreeStyle 293 Expression System (Invitrogen GmbH,Karlsruhe, Germany) according to the manufacturers protocol. After 3days cell culture supernatants of the transfectants were harvested andtested for the presence of the recombinant construct in an ELISA assay.Goat anti-human IgG, Fc-gamma fragment specific antibody (obtained fromJackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK) was dilutedin PBS to 5 μg/ml and coated with 100 μl per well onto a MaxiSorp96-well ELISA plate (Nunc GmbH & Co. KG, Wiesbaden, Germany) over nightat 4° C. Wells were washed with PBS with 0.05% Tween 20 (PBS/Tween andblocked with 3% BSA in PBS (bovine Albumin, fraction V, Sigma-AldrichChemie GmbH, Taufkirchen, Germany) for 60 minutes at room temperature(RT). Subsequently, wells were washed again PBS/Tween and then incubatedwith cell culture supernatants for 60 minutes at RT. After washing wellswere incubated with a peroxidase conjugated anti-His6 antibody (RocheDiagnostics GmbH, Roche Applied Science, Mannheim, Germany) diluted1:500 in PBS with 1% BSA for 60 minutes at RT. Subsequently, wells werewashed with 200 μl PBS/Tween and 100 μl of the SIGMAFAST OPD (SIGMAFASTOPD [o-Phenylenediamine dihydrochloride] substrate solution(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) was added according tothe manufacturers protocol. The reaction was stopped by adding 100 μl 1M H₂SO₄. Color reaction was measured on a PowerWaveX microplatespectrophotometer (BioTek Instruments, Inc., Winooski, Vt., USA) at 490nm and subtraction of background absorption at 620 nm. As shown in FIG.2 presence of the construct as compared to irrelevant supernatant ofmock-transfected HEK 293 cells used as negative control was clearlydetectable.

3.2. Binding Assay of Cross-Species Specific Single Chain Antibodies to1-27 CD3-Fc.

Binding of crude preparations of periplasmatically expressedcross-species specific single chain antibodies specific for CD3 epsilonto 1-27 CD3-Fc was tested in an ELISA assay. Goat anti-human IgG,Fc-gamma fragment specific antibody (Jackson ImmunoResearch Europe Ltd.,Newmarket, Suffolk, UK) was diluted in PBS to 5 μg/ml and coated with100 μl per well onto a MaxiSorp 96-well ELISA plate (Nunc GmbH & Co. KG,Wiesbaden, Germany) over night at 4° C. Wells were washed with PBS with0.05% Tween 20 (PBS/Tween and blocked with PBS with 3% BSA (bovineAlbumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)for 60 minutes at RT. Subsequently, wells were washed with PBS/Tween andincubated with supernatants of cells expressing the 1-27 CD3-Fcconstruct for 60 minutes at RT. Wells were washed with PBS/Tween andincubated with crude preparations of periplasmatically expressedcross-species specific single-chain antibodies as described above for 60minutes at room temperature. After washing with PBS/Tween wells wereincubated with peroxidase conjugated anti-Flag M2 antibody(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) diluted 1:10000 in PBSwith 1% BSA for 60 minutes at RT. Wells were washed with PBS/Tween andincubated with 100 μl of the SIGMAFAST OPD (OPD [o-Phenylenediaminedihydrochloride] substrate solution (Sigma-Aldrich Chemie GmbH,Taufkirchen, Germany) according to the manufacturers protocol. Colorreaction was stopped with 100 μl 1 M H₂SO₄ and measured on a PowerWaveXmicroplate spectrophotometer (BioTek Instruments, Inc., Winooski, Vt.,USA) at 490 nm and subtraction of background absorption at 620 nm.Strong binding of cross-species specific human single chain antibodiesspecific for CD3 epsilon to the 1-27 CD3-Fc construct compared to amurine anti CD3 single-chain antibody was observed (FIG. 3).

4. Generation of Recombinant Transmembrane Fusion Proteins of theN-Terminal Amino Acids 1-27 of CD3 Epsilon from Different Non-ChimpanzeePrimates Fused to EpCAM from Cynomolgus Monkey (1-27 CD3-EpCAM) 4.1.Cloning and Expression of 1-27 CD3-EpCAM

CD3 epsilon was isolated from different non-chimpanzee primates(marmoset, tamarin, squirrel monkey) and swine. The coding sequences ofthe 1-27 N-terminal amino acids of CD3 epsilon chain of the maturehuman, common marmoset (Callithrix jacchus), cottontop tamarin (Saguinusoedipus), common squirrel monkey (Saimiri sciureus) and domestic swine(Sus scrofa; used as negative control) fused to the N-terminus of Flagtagged cynomolgus EpCAM were obtained by gene synthesis according tostandard protocols. cDNA sequence and amino acid sequence of therecombinant fusion proteins are listed under SEQ ID NOs 231 to 240). Thegene synthesis fragments were designed as to contain first a BsrGl siteto allow fusion in correct reading frame with the coding sequence of a19 amino acid immunoglobulin leader peptide already present in thetarget expression vector, which is followed in frame by the codingsequence of the N-terminal 1-27 amino acids of the extracellular portionof the mature CD3 epsilon chains, which is followed in frame by thecoding sequence of a Flag tag and followed in frame by the codingsequence of the mature cynomolgus EpCAM transmembrane protein (FIG. 4).The gene synthesis fragments were also designed to introduce arestriction site at the end of the cDNA coding for the fusion protein.The introduced restriction sites BsrGI at the 5′ end and SalI at the 3′end, were utilized in the following cloning procedures. The genesynthesis fragments were then cloned via BsrGI and SalI into aderivative of the plasmid designated pEF DHFR (pEF-DHFR is described inMack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025), whichalready contained the coding sequence of the 19 amino acidimmunoglobulin leader peptide following standard protocols. Sequenceverified plasmids were used to transiently transfect 293-HEK cells usingthe MATra-A Reagent (IBA GmbH, Gottingen, Germany) and 12 μg of plasmidDNA for adherent 293-HEK cells in 175 ml cell culture flasks accordingto the manufacturers protocol. After 3 days of cell culture thetransfectants were tested for cell surface expression of the recombinanttransmembrane protein via an FACS assay according to standard protocols.For that purpose a number of 2.5×10⁵ cells were incubated with theanti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)at 5 μg/ml in PBS with 2% FCS. Bound antibody was detected with anR-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment, goatanti-mouse IgG, Fc-gamma fragment specific 1:100 in PBS with 2% FCS(Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Thesamples were measured on a FACScalibur (BD biosciences, Heidelberg,Germany). Expression of the Flag tagged recombinant transmembrane fusionproteins consisting of cynomolgus EpCAM and the 1-27 N-terminal aminoacids of the human, marmoset, tamarin, squirrel monkey and swine CD3epsilon chain respectively on transfected cells was clearly detectable(FIG. 5).

4.2. Binding of Cross-Species Specific Anti-CD3 Single Chain Antibodiesto the 1-27 CD3-EpCAM

Binding of crude preparations of periplasmatically expressedcross-species specific anti CD3 single-chain antibodies to the 1-27N-terminal amino acids of the human, marmoset, tamarin and squirrelmonkey CD3 epsilon chains respectively fused to cynomolgus Ep-CAM wastested in an FACS assay according to standard protocols. For thatpurpose a number of 2.5×10⁵ cells were incubated with crude preparationsof periplasmatically expressed cross-species specific anti CD3single-chain antibodies (preparation was performed as described aboveand according to standard protocols) and a single-chain murineanti-human CD3 antibody as negative control. As secondary antibody thePenta-His antibody (Qiagen GmbH, Hildesheim, Germany) was used at 5μg/ml in 50 μl PBS with 2% FCS. The binding of the antibody was detectedwith an R-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment,goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1:100 in PBSwith 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk,UK). The samples were measured on a FACScalibur (BD biosciences,Heidelberg, Germany). As shown in FIGS. 6 (A to E) binding of singlechain antibodies to the transfectants expressing the recombinanttransmembrane fusion proteins consisting of the 1-27 N-terminal aminoacids of CD3 epsilon of the human, marmoset, tamarin or squirrel monkeyfused to cynomolgus EpCAM was observed. No binding of cross-speciesspecific single chain antibodies was observed to a fusion proteinconsisting of the 1-27 N-terminal CD3 epsilon of swine fused tocynomolgus EpCAM used as negative control. Multi-primate cross-speciesspecificity of the anti-CD3 single chain antibodies was shown. Signalsobtained with the anti Flag M2 antibody and the cross-species specificsingle chain antibodies were comparable, indicating a strong bindingactivity of the cross-species specific single chain antibodies to theN-terminal amino acids 1-27 of CD3 epsilon.

5. Binding Analysis of Cross-Species Specific Anti-CD3 Single ChainAntibodies by Alanine-Scanning of Mouse Cells Transfected with the HumanCD3 Epsilon Chain and its Alanine Mutants 5.1. Cloning and Expression ofHuman Wild-Type CD3 Epsilon

The coding sequence of the human CD3 epsilon chain was obtained by genesynthesis according to standard protocols (cDNA sequence and amino acidsequence of the human CD3 epsilon chain are listed under SEQ ID NOs 242and 241). The gene synthesis fragment was designed as to contain a Kozaksite for eukaryotic expression of the construct and restriction sites atthe beginning and the end of the cDNA coding for human CD3 epsilon. Theintroduced restriction sites EcoRI at the 5′ end and SalI at the 3′ end,were utilized in the following cloning procedures. The gene synthesisfragment was then cloned via EcoRI and SalI into a plasmid designatedpEF NEO following standard protocols. pEF NEO was derived of pEF DHFR(Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) byreplacing the cDNA of the DHFR with the cDNA of the neomycin resistanceby conventional molecular cloning. A sequence verified plasmid was usedto transfect the murine T cell line EL4 (ATCC No. TIB-39) cultivated inRPMI with stabilized L-glutamine supplemented with 10% FCS, 1%penicillin/streptomycin, 1% HEPES, 1% pyruvate, 1% non-essential aminoacids (all Biochrom AG Berlin, Germany) at 37° C., 95% humidity and 7%CO₂. Transfection was performed with the SuperFect Transfection Reagent(Qiagen GmbH, Hilden, Germany) and 2 μg of plasmid DNA according to themanufacturer's protocol. After 24 hours the cells were washed with PBSand cultivated again in the aforementioned cell culture medium with 600μg/ml G418 for selection (PAA Laboratories GmbH, Pasching, Austria). 16to 20 days after transfection the outgrowth of resistant cells wasobserved. After additional 7 to 14 days cells were tested for expressionof human CD3 epsilon by FACS analysis according to standard protocols.2.5×10⁵ cells were incubated with anti-human CD3 antibody UCHT-1 (BDbiosciences, Heidelberg, Germany) at 5 μg/ml in PBS with 2% FCS. Thebinding of the antibody was detected with an R-Phycoerythrin-conjugatedaffinity purified F(ab′)2 fragment, goat anti-mouse IgG, Fc-gammafragment specific, diluted 1:100 in PBS with 2% FCS (JacksonImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). The samples weremeasured on a FACSCalibur (BD biosciences, Heidelberg, Germany).Expression of human wild-type CD3 on transfected EL4 cells is shown inFIG. 7.

5.2. Cloning and Expression of the Cross-Species Specific Anti-CD3Single Chain Antibodies as IgG1 Antibodies

In order to provide improved means of detection of binding of thecross-species specific single chain anti-CD3 antibodies H2C HLP, A2J HLPand E2M HLP were converted into IgG1 antibodies with murine IgG1 andhuman lambda constant regions. cDNA sequences coding for the heavy andlight chains of respective IgG antibodies were obtained by genesynthesis according to standard protocols. The gene synthesis fragmentsfor each specificity were designed as to contain first a Kozak site toallow eukaryotic expression of the construct, which is followed by an 19amino acid immunoglobulin leader peptide (SEQ ID NOs 244 and 243), whichis followed in frame by the coding sequence of the respective heavychain variable region or respective light chain variable region,followed in frame by the coding sequence of the heavy chain constantregion of murine IgG1 (SEQ ID NOs 246 and 245) or the coding sequence ofthe human lambda light chain constant region (SEQ ID NO 248 and 247),respectively. Restriction sites were introduced at the beginning and theend of the cDNA coding for the fusion protein. Restriction sites EcoRIat the 5′ end and SalI at the 3′ end were used for the following cloningprocedures. The gene synthesis fragments were cloned via EcoRI and SalIinto a plasmid designated pEF DHFR (Mack et al. Proc. Natl. Acad. Sci.USA 92 (1995) 7021-7025) for the heavy chain constructs and pEF ADA (pEFADA is described in Raum et al., Cancer Immunol Immunother., 50(3),(2001), 141-50) for the light chain constructs) according to standardprotocols. Sequence verified plasmids were used for co-transfection ofrespective light and heavy chain constructs in the FreeStyle 293Expression System (Invitrogen GmbH, Karlsruhe, Germany) according to themanufacturers protocol. After 3 days cell culture supernatants of thetransfectants were harvested and used for the alanine-scanningexperiment.

5.3. Cloning and Expression of Alanine Mutants of Human CD3 Epsilon forAlanine-Scanning

27 cDNA fragments coding for the human CD3 epsilon chain with anexchange of one codon of the wild-type sequence of human CD3 epsiloninto a codon coding for alanine (GCC) for each amino acid of amino acids1-27 of the extracellular domain of the mature human CD3 epsilon chainrespectively were obtained by gene synthesis. Except for the exchangedcodon the cDNA fragments were identical to the aforementioned humanwild-type CD3 cDNA fragment. Only one codon was replaced in eachconstruct compared to the human wild-type CD3 cDNA fragment describedabove. Restriction sites EcoRI and SalI were introduced into the cDNAfragments at identical positions compared to the wild-type construct.All alanine-scanning constructs were cloned into pEF NEO and sequenceverified plasmids were transfected into EL4 cells. Transfection andselection of transfectants was performed as described above. As result apanel of expressed constructs was obtained wherein the first amino acidof the human CD3 epsilon chain, glutamine (Q, Gln) at position 1 wasreplaced by alanine. The last amino acid replaced by alanine was thethreonine (T, Thr) at position 27 of mature human wild-type CD3 epsilon.For each amino acid between glutamine 1 and threonine 27 respectivetransfectants with an exchange of the wild-type amino acid into alaninewere generated.

5.4. Alanine-Scanning Experiment

Chimeric IgG antibodies as described in 5.2 and cross-species specificsingle chain antibodies specific for CD3 epsilon were tested inalanine-scanning experiment. Binding of the antibodies to the EL4 celllines transfected with the alanine-mutant constructs of human CD3epsilon as described in 5.3 was tested by FACS assay according tostandard protocols. 2.5×10⁵ cells of the respective transfectants wereincubated with 50 μl of cell culture supernatant containing the chimericIgG antibodies or with 50 μl of crude preparations of periplasmaticallyexpressed single-chain antibodies. For samples incubated with crudepreparations of periplasmatically expressed single-chain antibodies theanti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)was used as secondary antibody at 5 μg/ml in 50 μl PBS with 2% FCS. Forsamples incubated with the chimeric IgG antibodies a secondary antibodywas not necessary. For all samples the binding of the antibody moleculeswas detected with an R-Phycoerythrin-conjugated affinity purifiedF(ab′)2 fragment, goat anti-mouse IgG, Fc-gamma fragment specific,diluted 1:100 in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd.,Newmarket, Suffolk, UK). Samples were measured on a FACSCalibur (BDbiosciences, Heidelberg, Germany). Differential binding of chimeric IgGmolecules or cross-species specific single-chain antibodies to the EL4cell lines transfected with the alanine-mutants of human CD3 epsilon wasdetected. As negative control either an isotype control or a crudepreparation of a periplasmatically expressed single-chain antibody ofirrelevant specificity was used respectively. UCHT-1 antibody was usedas positive control for the expression level of the alanine-mutants ofhuman CD3 epsilon. The EL4 cell lines transfected with thealanine-mutants for the amino acids tyrosine at position 15, valine atposition 17, isoleucine at position 19, valine at position 24 or leucineat position 26 of the mature CD3 epsilon chain were not evaluated due tovery low expression levels (data not shown). Binding of thecross-species specific single chain antibodies and the single chainantibodies in chimeric IgG format to the EL4 cell lines transfected withthe alanine-mutants of human CD3 epsilon is shown in FIG. 8 (A-D) asrelative binding in arbitrary units with the geometric mean fluorescencevalues of the respective negative controls subtracted from allrespective geometric mean fluorescence sample values. To compensate fordifferent expression levels all sample values for a certain transfectantwere then divided through the geometric mean fluorescence value of theUCHT-1 antibody for the respective transfectant. For comparison with thewild-type sample value of a specificity all sample values of therespective specificity were finally divided through the wild-type samplevalue, thereby setting the wild-type sample value to 1 arbitrary unit ofbinding.

The calculations used are shown in detail in the following formula:

${{value\_ Sample}\left( {x,y} \right)} = \frac{{{Sample}\left( {x,y} \right)} - {{neg\_ Contr}.(x)}}{\begin{matrix}{\left( {{U\; C\; H\; T} - {1(x)} - {{neg\_ Contr}.(x)}} \right)*} \\\frac{{{WT}(y)} - {{neg\_ Contr}.({wt})}}{{U\; C\; H\; T} - {1({wt})} - {{neg\_ Contr}.({wt})}}\end{matrix}}$

In this equation value_Sample means the value in arbitrary units ofbinding depicting the degree of binding of a specific anti-CD3 antibodyto a specific alanine-mutant as shown in FIG. 8 (A-D), Sample means thegeometric mean fluorescence value obtained for a specific anti-CD3antibody assayed on a specific alanine-scanning transfectant, neg_Contr.means the geometric mean fluorescence value obtained for the negativecontrol assayed on a specific alanine-mutant, UCHT-1 means the geometricmean fluorescence value obtained for the UCHT-1 antibody assayed on aspecific alanine-mutant, WT means the geometric mean fluorescence valueobtained for a specific anti-CD3 antibody assayed on the wild-typetransfectant, x specifies the respective transfectant, y specifies therespective anti-CD3 antibody and wt specifies that the respectivetransfectant is the wild-type.

As can be seen in FIG. 8 (A-D) the IgG antibody A2J HLP showed apronounced loss of binding for the amino acids asparagine at position 4,threonine at position 23 and isoleucine at position 25 of the mature CD3epsilon chain. A complete loss of binding of IgG antibody A2J HLP wasobserved for the amino acids glutamine at position 1, aspartate atposition 2, glycine at position 3 and glutamate at position 5 of themature CD3 epsilon chain. IgG antibody E2M HLP showed a pronounced lossof binding for the amino acids asparagine at position 4, threonine atposition 23 and isoleucine at position 25 of the mature CD3 epsilonchain. IgG antibody E2M HLP showed a complete loss of binding for theamino acids glutamine at position 1, aspartate at position 2, glycine atposition 3 and glutamate at position 5 of the mature CD3 epsilon chain.IgG antibody H2C HLP showed an intermediate loss of binding for theamino acid asparagine at position 4 of the mature CD3 epsilon chain andit showed a complete loss of binding for the amino acids glutamine atposition 1, aspartate at position 2, glycine at position 3 and glutamateat position 5 of the mature CD3 epsilon chain. Single chain antibodyF12Q HLP showed an essentially complete loss of binding for the aminoacids glutamine at position 1, aspartate at position 2, glycine atposition 3 of the mature CD3 epsilon chain and glutamate at position 5of the mature CD3 epsilon chain.

6. Binding Analysis of the Cross-Species Specific Anti-CD3 BindingMolecule H2C HLP to the Human CD3 Epsilon Chain with and withoutN-Terminal His6 Tag Transfected into the Murine T Cell Line EL4

6.1. Cloning and Expression of the Human CD3 Epsilon Chain withN-Terminal Six Histidine Tag (His6 Tag)

A cDNA fragment coding for the human CD3 epsilon chain with a N-terminalHis6 tag was obtained by gene synthesis. The gene synthesis fragment wasdesigned as to contain first a Kozak site for eukaryotic expression ofthe construct, which is followed in frame by the coding sequence of a 19amino acid immunoglobulin leader peptide, which is followed in frame bythe coding sequence of a His6 tag which is followed in frame by thecoding sequence of the mature human CD3 epsilon chain (the cDNA andamino acid sequences of the construct are listed as SEQ ID NOs 256 and255). The gene synthesis fragment was also designed as to containrestriction sites at the beginning and the end of the cDNA. Theintroduced restriction sites EcoRI at the 5′ end and SalI at the 3′ end,were used in the following cloning procedures. The gene synthesisfragment was then cloned via EcoRI and SalI into a plasmid designatedpEF-NEO (as described above) following standard protocols. A sequenceverified plasmid was used to transfect the murine T cell line EL4.Transfection and selection of the transfectants were performed asdescribed above. After 34 days of cell culture the transfectants wereused for the assay described below.

6.2. Binding of the Cross-Species Specific Anti-CD3 Binding Molecule H2CHLP to the Human CD3 Epsilon Chain with and without N-Terminal His6 Tag

A chimeric IgG antibody with the binding specificity H2C HLP specificfor CD3 epsilon was tested for binding to human CD3 epsilon with andwithout N-terminal His6 tag. Binding of the antibody to the EL4 celllines transfected the His6-human CD3 epsilon and wild-type human CD3epsilon respectively was tested by an FACS assay according to standardprotocols. 2.5×10⁵ cells of the transfectants were incubated with 50 μlof cell culture supernatant containing the chimeric IgG antibody or 50μl of the respective control antibodies at 5 μg/ml in PBS with 2% FCS.As negative control an appropriate isotype control and as positivecontrol for expression of the constructs the CD3 specific antibodyUCHT-1 were used respectively. The binding of the antibodies wasdetected with a R-Phycoerythrin-conjugated affinity purified F(ab′)2fragment, goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1:100in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket,Suffolk, UK). Samples were measured on a FACSCalibur (BD biosciences,Heidelberg, Germany). Compared to the EL4 cell line transfected withwild-type human CD3 epsilon a clear loss of binding of the chimeric IgGwith binding specificity H2C HLP to human-CD3 epsilon with an N-terminalHis6 tag was detected. These results showed that a free N-terminus ofCD3 epsilon is essential for binding of the cross-species specificanti-CD3 binding specificity H2C HLP to the human CD3 epsilon chain(FIG. 9).

7. Cloning and Expression of the C-Terminal, Transmembrane and TruncatedExtracellular Domains of Human MCSP

The coding sequence of the C-terminal, transmembrane and truncatedextracellular domain of human MCSP (amino acids 1538-2322) was obtainedby gene synthesis according to standard protocols (cDNA sequence andamino acid sequence of the recombinant construct for expression of theC-terminal, transmembrane and truncated extracellular domain of humanMCSP (designated as human D3) are listed under SEQ ID NOs 250 and 249).The gene synthesis fragment was designed as to contain first a Kozaksite to allow eukaryotic expression of the construct followed by thecoding sequence of an 19 amino acid immunoglobulin leader peptidefollowed in frame by a FLAG tag, followed in frame by a sequencecontaining several restriction sites for cloning purposes and coding fora 9 amino acid artificial linker (SRTRSGSQL), followed in frame by thecoding sequence of the C-terminal, transmembrane and truncatedextracellular domain of human MCSP and a stop codon. Restriction siteswere introduced at the beginning and at the end of the DNA fragment. Therestriction sites EcoRI at the 5′ end and SalI at the 3′ end were usedin the following cloning procedures. The fragment was digested withEcoRI and SalI and cloned into pEF-DHFR (pEF-DHFR is described in Macket al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025) followingstandard protocols. A sequence verified plasmid was used to transfectCHO/dhfr-cells (ATCC No. CRL 9096). Cells were cultivated in RPMI 1640with stabilized glutamine, supplemented with 10% FCS, 1%penicillin/streptomycin (all obtained from Biochrom AG Berlin, Germany)and nucleosides from a stock solution of cell culture grade reagents(Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a finalconcentration of 10 μg/ml Adenosine, 10 μg/ml Deoxyadenosine and 10μg/ml Thymidine, in an incubator at 37° C., 95% humidity and 7% CO₂.Transfection was performed using the PolyFect Transfection Reagent(Qiagen GmbH, Hilden, Germany) and 5 μg of plasmid DNA according to themanufacturer's protocol. After cultivation for 24 hours cells werewashed once with PBS and cultivated again in RPMI 1640 with stabilizedglutamine and 1% penicillin/streptomycin. Thus the cell culture mediumdid not contain nucleosides and thereby selection was applied on thetransfected cells. Approximately 14 days after transfection theoutgrowth of resistant cells was observed. After an additional 7 to 14days the transfectants were tested for expression of the construct byFACS analysis. 2.5×10⁵ cells were incubated with 50 μl of ananti-Flag-M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)diluted to 5 μg/ml in PBS with 2% FCS. The binding of the antibody wasdetected with a R-Phycoerythrin-conjugated affinity purified F(ab′)2fragment, goat anti-mouse IgG, Fc-gamma fragment specific diluted 1:100in PBS with 2% FCS (ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK).The samples were measured on a FACScalibur (BD biosciences, Heidelberg,Germany).

8. Cloning and Expression of the C-Terminal, Transmembrane and TruncatedExtracellular Domains of Macaque MCSP

The cDNA sequence of the C-terminal, transmembrane and truncatedextracellular domains of macaque MCSP (designated as macaque D3) wasobtained by a set of three PCRs on macaque skin cDNA (Cat No.C1534218-Cy-BC; BioCat GmbH, Heidelberg, Germany) using the followingreaction conditions: 1 cycle at 94° C., 3 min., 40 cycles with 94° C.for 0.5 min., 52° C. for 0.5 min. and 72° C. for 1.75 min., terminalcycle of 72° C. for 3 min. The following primers were used:

forward primer: (SEQ ID No. 361) 5′-GATCTGGTCTACACCATCGAGC-3′ reverseprimer: (SEQ ID No. 362) 5′-GGAGCTGCTGCTGGCTCAGTGAGG-3′ forward primer:(SEQ ID No. 363) 5′-TTCCAGCTGAGCATGTCTGATGG-3′ reverse primer: (SEQ IDNo. 364) 5′-CGATCAGCATCTGGGCCCAGG-3′ forward primer: (SEQ ID No. 365)5′-GTGGAGCAGTTCACTCAGCAGGACC-3′ reverse primer: (SEQ ID No. 366)5′-GCCTTCACACCCAGTACTGGCC-3′

Those PCRs generated three overlapping fragments (A: 1-1329, B:1229-2428, C: 1782-2547) which were isolated and sequenced according tostandard protocols using the PCR primers and thereby provided a 2547 bpportion of the cDNA sequence of macaque MCSP (the cDNA sequence andamino acid sequence of this portion of macaque MCSP are listed under SEQID NOs 252 and 251) from 74 bp upstream of the coding sequence of theC-terminal domain to 121 bp downstream of the stop codon. Another PCRusing the following reaction conditions: 1 cycle at 94° C. for 3 min, 10cycles with 94° C. for 1 min, 52° C. for 1 min and 72° C. for 2.5 min,terminal cycle of 72° C. for 3 min was used to fuse the PCR products ofthe aforementioned reactions A and B. The following primers are used:

forward primer: (SEQ ID No. 367)5′-tcccgtacgagatctggatcccaattggatggcggactcgtgctg ttctcacacagagg-3′reverse primer: (SEQ ID No. 368)5′-agtgggtcgactcacacccagtactggccattcttaagggca ggg-3′

The primers for this PCR were designed to introduce restriction sites atthe beginning and at the end of the cDNA fragment coding for theC-terminal, transmembrane and truncated extracellular domains of macaqueMCSP. The introduced restriction sites MfeI at the 5′ end and SalI atthe 3′ end, were used in the following cloning procedures. The PCRfragment was then cloned via MfeI and SalI into a Bluescript plasmidcontaining the EcoRI/MfeI fragment of the aforementioned plasmidpEF-DHFR (pEF-DHFR is described in Raum et al. Cancer Immunol Immunother50 (2001) 141-150) by replacing the C-terminal, transmembrane andtruncated extracellular domains of human MCSP. The gene synthesisfragment contained the coding sequences of the immunoglobulin leaderpeptide and the Flag tag as well as the artificial linker (SRTRSGSQL) inframe to the 5′ end of the cDNA fragment coding for the C-terminal,transmembrane and truncated extracellular domains of macaque MCSP. Thisvector was used to transfect CHO/dhfr-cells (ATCC No. CRL 9096). Cellswere cultivated in RPMI 1640 with stabilized glutamine supplemented with10% FCS, 1% penicillin/streptomycin (all from Biochrom AG Berlin,Germany) and nucleosides from a stock solution of cell culture gradereagents (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) to a finalconcentration of 10 μg/ml Adenosine, 10 μg/ml Deoxyadenosine and 10μg/ml Thymidine, in an incubator at 37° C., 95% humidity and 7% CO2.Transfection was performed with PolyFect Transfection Reagent (QiagenGmbH, Hilden, Germany) and 5 μg of plasmid DNA according to themanufacturer's protocol. After cultivation for 24 hours cells werewashed once with PBS and cultivated again in RPMI 1640 with stabilizedglutamine and 1% penicillin/streptomycin. Thus the cell culture mediumdid not contain nucleosides and thereby selection was applied on thetransfected cells. Approximately 14 days after transfection theoutgrowth of resistant cells is observed. After an additional 7 to 14days the transfectants were tested for expression of the recombinantconstruct via FACS. 2.5×10⁵ cells were incubated with 50 μl of ananti-Flag-M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)diluted to 5 μg/ml in PBS with 2% FCS. Bound antibody was detected witha R-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment, goatanti-mouse IgG, Fc-gamma fragment specific, diluted 1:100 in PBS with 2%FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK).Samples were measured on a FACScalibur (BD biosciences, Heidelberg,Germany).

9. Generation and Characterisation of MCSP and CD3 Cross-SpeciesSpecific Bispecific Single Chain Molecules

Bispecific single chain antibody molecules each comprising a bindingdomain cross-species specific for human and non-chimpanzee primate CD3epsilon as well as a binding domain cross-species-specific for human andnon-chimpanzee primate MCSP, are designed as set out in the followingTable 1:

TABLE 1 Formats of MCSP and CD3 cross-species specific bispecific singlechain antibodies SEQ ID Formats of protein constructs (nucl/prot) (N →C) 190/189 MCSP-G4 HL × H2C HL 192/191 MCSP-G4 HL × F12Q HL 194/193MCSP-G4 HL × I2C HL 196/195 MCSP-G4 HLP × F6A HLP 198/197 MCSP-G4 HLP ×H2C HLP 202/201 MCSP-G4 HLP × G4H HLP 206/205 MCSP-G4 HLP × E1L HLP208/207 MCSP-G4 HLP × E2M HLP 212/211 MCSP-G4 HLP × F12Q HL 214/213MCSP-G4 HLP × I2C HL 216/215 MCSP-D2 HL × H2C HL 218/217 MCSP-D2 HL ×F12Q HL 220/219 MCSP-D2 HL × I2C HL 222/221 MCSP-D2 HLP × H2C HLP224/223 MCSP-F9 HL × H2C HL 226/225 MCSP-F9 HLP × H2C HLP 228/227MCSP-F9 HLP × G4H HLP 318/317 MCSP-A9 HL × H2C HL 320/319 MCSP-A9 HL ×F12Q HL 322/321 MCSP-A9 HL × I2C HL 324/323 MCSP-C8 HL × I2C HL 328/327MCSP-B7 HL × I2C HL 326/325 MCSP-B8 HL × I2C HL 330/329 MCSP-G8 HL × I2CHL 332/331 MCSP-D5 HL × I2C HL 334/333 MCSP-F7 HL × I2C HL 336/335MCSP-G5 HL × I2C HL 338/337 MCSP-F8 HL × I2C HL 340/339 MCSP-G10 HL ×I2C HL

The aforementioned constructs containing the variable heavy-chain (VH)and variable light-chain (VL) domains cross-species specific for humanand macaque MCSP D3 and the VH and VL domains cross-species specific forhuman and macaque CD3 were obtained by gene synthesis. The genesynthesis fragments were designed as to contain first a Kozak site foreukaryotic expression of the construct, followed by a 19 amino acidimmunoglobulin leader peptide, followed in frame by the coding sequenceof the respective bispecific single chain antibody molecule, followed inframe by the coding sequence of a histidine₆-tag and a stop codon. Thegene synthesis fragment was also designed as to introduce suitable N-and C-terminal restriction sites. The gene synthesis fragment was clonedvia these restriction sites into a plasmid designated pEF-DHFR (pEF-DHFRis described in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150)according to standard protocols (Sambrook, Molecular Cloning; ALaboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press,Cold Spring Harbour, N.Y. (2001)). The constructs were transfectedstably or transiently into DHFR-deficient CHO-cells (ATCC No. CRL 9096)by electroporation or alternatively into HEK 293 (human embryonal kidneycells, ATCC Number: CRL-1573) in a transient manner according tostandard protocols.

Eukaryotic protein expression in DHFR deficient CHO cells was performedas described by Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566.Gene amplification of the constructs was induced by addition ofincreasing concentrations of methothrexate (MTX) up to finalconcentrations of 20 nM MTX. After two passages of stationary culturethe cells were grown in roller bottles with nucleoside-free HyQ PF CHOliquid soy medium (with 4.0 mM L-Glutamine with 0.1% Pluronic F-68;HyClone) for 7 days before harvest. The cells were removed bycentrifugation and the supernatant containing the expressed protein isstored at −20° C.

Äkta® Explorer System (GE Health Systems) and Unicorn® Software wereused for chromatography. Immobilized metal affinity chromatography(“IMAC”) was performed using a Fractogel EMD Chelate® (Merck) which wasloaded with ZnCl₂ according to the protocol provided by themanufacturer. The column was equilibrated with buffer A (20 mM sodiumphosphate buffer pH 7.2, 0.1 M NaCl) and the cell culture supernatant(500 ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.The column was washed with buffer A to remove unbound sample. Boundprotein was eluted using a two step gradient of buffer B (20 mM sodiumphosphate buffer pH 7.2, 0.1 M NaCl, 0.5 M Imidazole) according to thefollowing:

Step 1: 20% buffer B in 6 column volumesStep 2: 100% buffer B in 6 column volumes

Eluted protein fractions from step 2 were pooled for furtherpurification. All chemicals are of research grade and purchased fromSigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration chromatography was performed on a HiLoad 16/60 Superdex200 prep grade column (GE/Amersham) equilibrated with Equi-buffer (25 mMCitrate, 200 mM Lysine, 5% Glycerol, pH 7.2). Eluted protein samples(flow rate 1 ml/min) were subjected to standard SDS-PAGE and WesternBlot for detection. Prior to purification, the column was calibrated formolecular weight determination (molecular weight marker kit, Sigma MWGF-200). Protein concentrations were determined using OD280 nm.

Purified bispecific single chain antibody protein was analyzed in SDSPAGE under reducing conditions performed with pre-cast 4-12% Bis Trisgels (Invitrogen). Sample preparation and application were performedaccording to the protocol provided by the manufacturer. The molecularweight was determined with MultiMark protein standard (Invitrogen). Thegel was stained with colloidal Coomassie (Invitrogen protocol). Thepurity of the isolated protein is >95% as determined by SDS-PAGE.

The bispecific single chain antibody has a molecular weight of about 52kDa under native conditions as determined by gel filtration in phosphatebuffered saline (PBS). All constructs were purified according to thismethod.

Western Blot was performed using an Optitran® BA-S83 membrane and theInvitrogen Blot Module according to the protocol provided by themanufacturer. For detection of the bispecific single chain antibodyprotein antibodies an anti-His Tag antibody was used (Penta His,Qiagen). A Goat-anti-mouse Ig antibody labeled with alkaline phosphatase(AP) (Sigma) was used as secondary antibody and BCIP/NBT (Sigma) assubstrate. A single band was detected at 52 kD corresponding to thepurified bispecific single chain antibody.

Alternatively, constructs were transiently expressed in DHFR deficientCHO cells. In brief, 4×105 cells per construct were cultivated in 3 mlRPMI 1640 all medium with stabilized glutamine supplemented with 10%fetal calf serum, 1% penicillin/streptomycin and nucleosides from astock solution of cell culture grade reagents (Sigma-Aldrich ChemieGmbH, Taufkirchen, Germany) to a final concentration of 10 μg/mlAdenosine, 10 μg/ml Deoxyadenosine and 10 μg/ml Thymidine, in anincubator at 37° C., 95% humidity and 7% CO2 one day beforetransfection. Transfection was performed with Fugene 6 TransfectionReagent (Roche, #11815091001) according to the manufacturer's protocol.94 μl OptiMEM medium (Invitrogen) and 6 μl Fugene 6 are mixed andincubated for 5 minutes at room temperature. Subsequently, 1.5 μg DNAper construct were added, mixed and incubated for 15 minutes at roomtemperature. Meanwhile, the DHFR deficient CHO cells were washed with1×PBS and resuspended in 1.5 ml RPMI 1640 all medium. The transfectionmix was diluted with 600 μl RPMI 1640 all medium, added to the cells andincubated overnight at 37° C., 95% humidity and 7% CO2. The day aftertransfection the incubation volume of each approach was extended to 5 mlRPMI 1640 all medium. Supernatant was harvested after 3 days ofincubation.

10. Flow Cytometric Binding Analysis of the MCSP and CD3 Cross-SpeciesSpecific Bispecific Antibodies

In order to test the functionality of the cross-species specificbispecific antibody constructs regarding the capability to bind to humanand macaque MCSP D3 and CD3, respectively, a FACS analysis wasperformed. For this purpose CHO cells transfected with human MCSP D3 (asdescribed in Example 7) and the human CD3 positive T cell leukemia cellline HPB-ALL (DSMZ, Braunschweig, ACC483) were used to test the bindingto human antigens. The binding reactivity to macaque antigens was testedby using the generated macaque MCSP D3 transfectant (described inExample 8) and a macaque T cell line 4119LnPx (kindly provided by Prof.Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; publishedin Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61).200.000 cells of the respective cell lines were incubated for 30 min onice with 50 μl of the purified protein of the cross-species specificbispecific antibody constructs (2 μg/ml) or cell culture supernatant oftransfected cells expressing the cross-species specific bispecificantibody constructs. The cells were washed twice in PBS with 2% FCS andbinding of the construct was detected with a murine anti-His antibody(Penta His antibody; Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS).After washing, bound anti-His antibodies were detected with an Fcgamma-specific antibody (Dianova) conjugated to phycoerythrin, diluted1:100 in PBS with 2% FCS. Supernatant of untransfected CHO cells wasused as negative control for binding to the T cell lines. A single chainconstruct with irrelevant target specificity was used as negativecontrol for binding to the MCSP-D3 transfected CHO cells.

Flow cytometry was performed on a FACS-Calibur apparatus; the CellQuestsoftware was used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

The bispecific binding of the single chain molecules listed above, whichare cross-species specific for MCSP D3 and cross-species specific forhuman and macaque CD3 was clearly detectable as shown in FIGS. 10, 11,12 and 39. In the FACS analysis all constructs showed binding to CD3 andMCSP D3 as compared to the respective negative controls. Cross-speciesspecificity of the bispecific antibodies to human and macaque CD3 andMCSP D3 antigens was demonstrated.

11. Bioactivity of MCSP and CD3 Cross-Species Specific Bispecific SingleChain Antibodies

Bioactivity of the generated bispecific single chain antibodies wasanalyzed by chromium 51 (⁵¹Cr) release in vitro cytotoxicity assaysusing the MCSP D3 positive cell lines described in Examples 7 and 8. Aseffector cells stimulated human CD4/CD56 depleted PBMC, stimulated humanPBMC or the macaque T cell line 4119LnPx are used as specified in therespective figures.

Generation of the stimulated CD4/CD56 depleted PBMC was performed asfollows: Coating of a Petri dish (145 mm diameter, Greiner bio-one GmbH,Kremsmünster) was carried out with a commercially available anti-CD3specific antibody (e.g. OKT3, Othoclone) in a final concentration of 1μg/ml for 1 hour at 37° C. Unbound protein was removed by one washingstep with PBS. The fresh PBMC were isolated from peripheral blood (30-50ml human blood) by Ficoll gradient centrifugation according to standardprotocols. 3-5×10⁷ PBMC were added to the precoated petri dish in 120 mlof RPMI 1640 with stabilized glutamine/10% FCS/IL-2 20 U/ml (Proleukin,Chiron) and stimulated for 2 days. On the third day the cells werecollected and washed once with RPMI 1640. IL-2 was added to a finalconcentration of 20 U/ml and the cells were cultivated again for one dayin the same cell culture medium as above. By depletion of CD4+ T cellsand CD56+ NK cells according to standard protocols CD8+ cytotoxic Tlymphocytes (CTLs) were enriched.

Target cells were washed twice with PBS and labelled with 11.1 MBq ⁵¹Crin a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37° C.Subsequently the labelled target cells were washed 3 times with 5 mlRPMI and then used in the cytotoxicity assay. The assay was performed ina 96 well plate in a total volume of 250 μl supplemented RPMI (as above)with an E:T ratio 10:1. 1 μg/ml of the cross-species specific bispecificsingle chain antibody molecules and 20 threefold dilutions thereof wereapplied. If using supernatant containing the cross-species specificbispecific single chain antibody molecules, 21 two- and 20 threefolddilutions thereof were applied for the macaque and the humancytotoxicity assay, respectively. The assay time was 18 hours andcytotoxicity was measured as relative values of released chromium in thesupernatant related to the difference of maximum lysis (addition ofTriton-X) and spontaneous lysis (without effector cells). Allmeasurements were done in quadruplicates. Measurement of chromiumactivity in the supernatants was performed with a Wizard 3″ gammacounter (Perkin Elmer Life Sciences GmbH, Köln, Germany). Analysis ofthe experimental data was performed with Prism 4 for Windows (version4.02, GraphPad Software Inc., San Diego, Calif., USA). Sigmoidal doseresponse curves typically have R² values >0.90 as determined by thesoftware. EC₅₀ values calculated by the analysis program were used forcomparison of bioactivity.

As shown in FIGS. 13 to 17 and 40, all of the generated cross-speciesspecific bispecific single chain antibody constructs demonstratecytotoxic activity against human MCSP D3 positive target cells elicitedby stimulated human CD4/CD56 depleted PBMC or stimulated PBMC andagainst macaque MCSP D3 positive target cells elicited by the macaque Tcell line 4119LnPx.

12. Plasma Stability of MCSP and CD3 Cross-Species Specific BispecificSingle Chain Antibodies

Stability of the generated bispecific single chain antibodies in humanplasma was analyzed by incubation of the bispecific single chainantibodies in 50% human Plasma at 37° C. and 4° C. for 24 hours andsubsequent testing of bioactivity. Bioactivity was studied in a chromium51 (⁵¹Cr) release in vitro cytotoxicity assay using a MCSP positive CHOcell line (expressing MCSP as cloned according to example 14 or 15) astarget and stimulated human CD8 positive T cells as effector cells.

EC₅₀ values calculated by the analysis program as described above wereused for comparison of bioactivity of bispecific single chain antibodiesincubated with 50% human plasma for 24 hours at 37° C. and 4° C.respectively with bispecific single chain antibodies without addition ofplasma or mixed with the same amount of plasma immediately prior to theassay.

As shown in FIG. 18 and Table 2 the bioactivity of the G4 H-L×I2C H-L,G4 H-L×H2C H-L and G4 H-L×F12Q H-L bispecific antibodies was notsignificantly reduced as compared with the controls without the additionof plasma or with addition of plasma immediately before testing ofbioactivity.

TABLE 2 bioactivity of the bispecific antibodies without or with theaddition of Plasma Construct Without plasma With plasma Plasma 37° C.Plasma 4° C. G4 H-L × 300 796 902 867 I2C H-L G4 H-L × 496 575 2363 1449H2C H-L G4 H-L × 493 358 1521 1040 F12Q H-L

13. Redistribution of Circulating T Cells in the Absence of CirculatingTarget Cells by First Exposure to CD3 Binding Molecules Directed atConventional i.e. Context Dependent CD3 Epitopes is a Major Risk Factorfor Adverse Events Related to the Initiation of Treatment

T Cell Redistribution in Patients with B-Cell Non-Hodgkin-Lymphoma(B-NHL) Following Initiation of Treatment with the Conventional CD3Binding Molecule

A conventional CD19×CD3 binding molecules is a CD3 binding molecule ofthe bispecific tandem scFv format (Loffler (2000, Blood, Volume 95,Number 6) or WO 99/54440). It consists of two different binding portionsdirected at (i) CD19 on the surface of normal and malignant human Bcells and (ii) CD3 on human T cells. By crosslinking CD3 on T cells withCD19 on B cells, this construct triggers the redirected lysis of normaland malignant B cells by the cytotoxic activity of T cells. The CD3epitope recognized by such a conventional CD3 binding molecule islocalized on the CD3 epsilon chain, where it only takes the correctconformation if it is embedded within the rest of the epsilon chain andheld in the right position by heterodimerization of the epsilon chainwith either the CD3 gamma or delta chain. Interaction of this highlycontext dependent epitope with a conventional CD3 binding molecule (seee.g. Loffler (2000, Blood, Volume 95, Number 6) or WO 99/54440)—evenwhen it occurs in a purely monovalent fashion and without anycrosslinking—can induce an allosteric change in the conformation of CD3leading to the exposure of an otherwise hidden proline-rich regionwithin the cytoplasmic domain of CD3 epsilon. Once exposed, theproline-rich region can recruit the signal transduction molecule Nck2,which is capable of triggering further intracellular signals. Althoughthis is not sufficient for full T cell activation, which definitelyrequires crosslinking of several CD3 molecules on the T cell surface,e.g. by crosslinking of several anti-CD3 molecules bound to several CD3molecules on a T cell by several CD19 molecules on the surface of a Bcell, pure monovalent interaction of conventional CD3 binding moleculesto their context dependent epitope on CD3 epsilon is still not inert forT cells in terms of signalling. Without being bound by theory,monovalent conventional CD3 binding molecules (known in the art) mayinduce some T cell reactions when infused into humans even in thosecases where no circulating target cells are available for CD3crosslinking. An important T cell reaction to the intravenous infusionof monovalent conventional CD19×CD3 binding molecule into B-NHL patientswho have essentially no circulating CD19-positive B cells is theredistribution of T cells after start of treatment. It has been found ina phase I clinical trial that this T cell reaction occurs during thestarting phase of intravenous CD19×CD3 binding molecule infusion in allindividuals without circulating CD19-positive target B cells essentiallyindependent of the CD19×CD3 binding molecule dose (FIG. 19). However,sudden increases in CD19×CD3 binding molecule exposure have been foundto trigger virtually the same redistributional T cell reaction in thesepatients as the initial exposure of T cells to CD19×CD3 binding moleculeat treatment start (FIG. 20 A) and even gradual increases in CD19×CD3binding molecule exposure still can have redistributional effects oncirculating T cells (FIG. 21). Moreover, it has been found that thisessentially dose-independent redistributional T cell reaction in theabsence of circulating target cells as triggered by conventional CD3binding molecules like the CD19×CD3 binding molecule (e.g. disclosed inWO 99/54440) in 100% of all treated individuals is a major risk factorfor adverse events related to the initiation of treatment.

According to the study protocol, patients with relapsed histologicallyconfirmed indolent B-cell Non-Hodgkin-Lymphoma (B-NHL) including mantlecell lymphoma were recruited in an open-label, multi-center phase Iinterpatient dose-escalation trial. The study protocol was approved bythe independent ethics committees of all participating centers and sentfor notification to the responsible regulatory authority. Measurabledisease (at least one lesion ≧1.5 cm) as documented by CT scan wasrequired for inclusion into the study. Patients received conventionalCD19×CD3 binding molecule by continuous intravenous infusion with aportable minipump system over four weeks at constant flow rate (i.e.dose level). Patients were hospitalized during the first two weeks oftreatment before they were released from the hospital and continuedtreatment at home. Patients without evidence of disease progressionafter four weeks were offered to continue treatment for further fourweeks. So far six different dose levels were tested without reaching amaximum tolerated dose (MTD): 0.5, 1.5, 5, 15, 30 and 60 μg/m²/24 h.Cohorts consisted of three patients each if no adverse events defined bythe study protocol as DLT (dose limiting toxicity) were observed. Incase of one DLT among the first three patients the cohort was expandedto six patients, which—in the absence of a second DLT—allowed furtherdose escalation. Accordingly, dose levels without DLT in cohorts with 3patients or with one DLT in cohorts with 6 patients were regarded assafe. Study treatment was stopped in all patients who developed a DLT.At 15 and 30 μg/m²/24 h different modes of treatment initiation duringthe first 24 h were tested in several additional cohorts: (i) Stepwiseincrease after 5 μg/m²/24 h for the first 24 h to 15 μg/m²/24 hmaintenance dose (patient cohort 15-step), (ii) even continuous increaseof flow-rate from almost zero to 15 or 30 μg/m²/24 h (patient cohorts15-ramp and 30-ramp) and (iii) start with the maintenance dose from thevery beginning (patient cohorts 15-flat, 30-flat and 60-flat). Patientcohorts at dose levels 0.5, 1.5 and 5 μg/m²/24 h were all started withthe maintenance dose from the very beginning (i.e. flat initiation).

Time courses of absolute B- and T-cell counts in peripheral blood weredetermined by four color FACS analysis as follows:

Collection of Blood Samples and Routine Analysis

In patient cohorts 15-ramp, 15-flat, 30-ramp, 30-flat and 60-flat bloodsamples (6 ml) were obtained before and 0.75, 2, 6, 12, 24, 30, 48 hoursafter start of CD19×CD3 binding molecule (as disclosed in WO 99/54440)infusion as well as on treatment days 8, 15, 17, 22, 24, 29, 36, 43, 50,57 and 4 weeks after end of conventional CD19×CD3 binding moleculeinfusion using EDTA-containing Vacutainer™ tubes (Becton Dickinson)which were shipped for analysis at 4° C. In patient cohorts 15-stepblood samples (6 ml) were obtained before and 6, 24, 30, 48 hours afterstart of CD19×CD3 binding molecule infusion as well as on treatment days8, 15, 22, 29, 36, 43, 50, 57 and 4 weeks after end of CD19×CD3 bindingmolecule infusion. At dose levels 0.5, 1.5 and 5 μg/m²/24 h bloodsamples (6 ml) were obtained before and 6, 24, 48 hours after start ofCD19×CD3 binding molecule infusion as well as on treatment days 8, 15,22, 29, 36, 43, 50, 57 and 4 weeks after end of CD19×CD3 bindingmolecule infusion. In some cases slight variations of these time pointsoccurred for operational reasons. FACS analysis of lymphocytesubpopulations was performed within 24-48 h after blood samplecollection. Absolute numbers of leukocyte subpopulations in the bloodsamples were determined through differential blood analysis on aCoulterCounter™ (Coulter).

Isolation of PBMC from Blood Samples

PBMC (peripheral blood mononuclear cells) isolation was performed by anadapted Ficoll™ gradient separation protocol. Blood was transferred atroom temperature into 10 ml Leucosep™ tubes (Greiner) pre-loaded with 3ml Biocoll™ solution (Biochrom). Centrifugation was carried out in aswing-out rotor for 15 min at 1700×g and 22° C. without deceleration.The PBMC above the Biocoll™ layer were isolated, washed once with FACSbuffer (PBS/2% FBS [Foetal Bovine Serum; Biochrom]), centrifuged andresuspended in FACS buffer. Centrifugation during all wash steps wascarried out in a swing-out rotor for 4 min at 800×g and 4° C. Ifnecessary, lysis of erythrocytes was performed by incubating theisolated PBMC in 3 ml erythrocyte lysis buffer (8.29 g NH₄Cl, 1.00 gKHCO₃, 0.037 g EDTA, ad 1.0 l H₂O_(bidest), pH 7.5) for 5 min at roomtemperature followed by a washing step with FACS buffer.

Staining of PBMC with Fluorescence-Labeled Antibodies Against CellSurface Molecules

Monoclonal antibodies were obtained from Invitrogen (¹Cat. No. MHCD1301,²Cat. No. MHCD1401), Dako (⁵Cat. No. C7224) or Becton Dickinson (³Cat.No. 555516, ⁴Cat. No. 345766) used according to the manufacturers'recommendations. 5×10⁵-1×10⁶ cells were stained with the followingantibody combination: anti-CD13¹/anti-CD14² (FITC)×anti-CD56³(PE)×anti-CD3⁴ (PerCP)×anti-CD19⁵ (APC). Cells were pelleted in V-shaped96 well multititer plates (Greiner) and the supernatant was removed.Cell pellets were resuspended in a total volume of 100 μl containing thespecific antibodies diluted in FACS buffer. Incubation was carried outin the dark for 30 min at 4° C. Subsequently, samples were washed twicewith FACS buffer and cell pellets were resuspended in FACS buffer forflowcytometric analysis.

Flowcytometric Detection of Stained Lymphocytes by FACS

Data collection was performed with a 4 color BD FACSCalibur™ (BectonDickinson). For each measurement 1×10⁴ cells of defined lymphocytesubpopulations were acquired. Statistical analysis was performed withthe program CellQuest Pro™ (Becton Dickinson) to obtain lymphocytesubpopulation percentages and to classify cell surface moleculeexpression intensity. Subsequently, percentages of single lymphocytesubsets related to total lymphocytes (i.e. B plus T plus NK cellsexcluding any myeloid cells via CD13/14-staining) as determined by FACSwere correlated with the lymphocyte count from the differential bloodanalysis to calculate absolute cell numbers of T cells (CD3⁺, CD56⁻,CD13/14⁻) and B cells (CD19⁺, CD13/14⁻).

T cell redistribution during the starting phase of conventional CD19×CD3binding molecule (e.g. disclosed in WO 99/54440) treatment in all thosepatients who had essentially no circulating CD19-positive B cells attreatment start is shown in (FIG. 19). For comparison, a representativeexample of T cell redistribution during the starting phase of CD19×CD3binding molecule treatment in a patient with a significant number ofcirculating CD19-positive B cells is shown in FIG. 22.

In both cases (i.e. essentially no or many circulating B cells)circulating T cell counts rapidly decrease upon treatment start.However, in the absence of circulating B cells T cells tend to returninto the circulating blood very early, while the return of T cells intothe circulating blood of those patients who have a significant number ofcirculating B cells at treatment start is usually delayed until thesecirculating B cells are depleted. Thus, the T cell redistributionpatterns mainly differ in the kinetics of T cell reappearance in thecirculating blood.

Assessment of efficacy based on CT scan was carried out by centralreference radiology after 4 weeks of treatment and in patients receivingadditional 4 weeks also after 8 weeks of treatment plus in all casesfour weeks after end of treatment. Disappearance and/or normalization insize of all known lesions (including an enlarged spleen) plus clearanceof bone marrow from lymphoma cells in cases of bone marrow infiltrationwas counted as complete response (CR). Reduction by at least 50% frombaseline of the sum of products of the two biggest diameters (SPD) ofeach predefined target lesion was defined as partial response (PR); areduction by at least 25% was regarded a minimal response (MR).Progressive disease (PD) was defined as ≧50% increase of SPD frombaseline. SPD deviations from baseline between +50% and −25% wereregarded as stable disease (SD).

Patient demographics, doses received and clinical outcome in 34 patientsare summarized in Table 3. Clinical anti-tumor activity of the CD19×CD3binding molecule was clearly dose dependent: Consistent depletion ofcirculating CD19-positive B (lymphoma) cell from peripheral blood wasobserved from 5 μg/m²/24 h onwards. At 15 μg/m²/24 h and 30 μg/m²/24 hfirst objective clinical responses (PRs and CRs) were recorded as wellas cases of partial and complete elimination of B lymphoma cells frominfiltrated bone marrow. Finally, at 60 μg/m²/24 h the response rateincreased to 100% (PRs and CRs) and bone marrow clearance from Blymphoma cells was complete in all evaluable cases.

The CD19×CD3 binding molecule was well tolerated by the majority ofpatients. Most frequent adverse events of grades 1-4 in 34 patients,regardless of causality are summarized in Table 4. CD19×CD3 bindingmolecule-related adverse events usually were transient and fullyreversible. In particular, there were 2 patients (patients #19 and #24in Table 3) essentially without circulating CD19-positive B cells whosetreatment was stopped early because of CNS adverse events (leadsymptoms: confusion and disorientation) related to repeated T cellredistribution during the starting phase of CD19×CD3 binding moleculeinfusion.

One of these patients (#19) was in cohort 15-step. He received 5μg/m²/24 h CD19×CD3 binding molecule for the first 24 h followed bysudden increase to 15 μg/m²/24 h maintenance dose. The corresponding Tcell redistribution pattern shows that circulating T cell counts rapidlydecreased upon start of infusion at 5 μg/m²/24 h followed by earlyreappearance of T cells in the circulating blood essentially withoutcirculating CD19-positive B cells. As a consequence, the peripheral Tcell counts had fully recovered when the CD19×CD3 binding molecule dosewas increased after 24 h from 5 to 15 μg/m²/24 h. Therefore the dosestep could trigger a second episode of T cell redistribution as shown inFIG. 20 A. This repeated T cell redistribution was related with CNS sideeffects (lead symptoms: confusion and disorientation) in this patient,which led to the stop of infusion. The relationship between repeated Tcell redistribution and such CNS adverse events was also observed inprevious phase I clinical trials in B-NHL patients who received CD19×CD3binding molecule (e.g. disclosed in WO 99/54440) as repeated bolusinfusion for 2 to 4 hours each usually followed by 2 days of treatmentfree interval (FIG. 20 B). Every single bolus infusion triggered oneepisode of T cell redistribution consisting of a fast decrease incirculating T cell counts and T cell recovery prior to the next bolusinfusion. In total, CNS adverse events related to repeated T cellredistribution were observed in 5 out of 21 patients. FIG. 20 B showsthe representative example of one patient from the bolus infusiontrials, who developed CNS symptoms after the third episode of T cellredistribution. Typically, patients with CNS adverse events in the bolusinfusion trials also had low circulating B cell counts.

The second patient (#24) from the continuous infusion trial, whosetreatment was stopped early because of CNS adverse events (leadsymptoms: confusion and disorientation) related to repeated T cellredistribution during the starting phase of CD19×CD3 binding moleculeinfusion, was in cohort 15-flat. By mistake, this patient received anCD19×CD3 binding molecule infusion without additional HSA as requiredfor stabilization of the drug. The resulting uneven drug flow triggeredrepeated episodes of T cell redistribution instead of only one (FIG. 23A) with the consequence that the infusion had to be stopped because ofdeveloping CNS symptoms. Yet, when the same patient was restartedcorrectly with CD19×CD3 binding molecule solution containing additionalHSA for drug stabilization (e.g. disclosed in WO 99/54440), no repeatedT cell redistribution was observed and the patient did not again developany CNS symptoms (FIG. 23 B). Because this patient also had essentiallyno circulating B cells, the circulating T cells could react with fastredistribution kinetics even to subtle changes in drug exposure asobserved. The CNS adverse events related to T cell redistribution inpatients who have essentially no circulating target cells can beexplained by a transient increase of T cell adhesiveness to theendothelial cells followed by massive simultaneous adhesion ofcirculating T cells to the blood vessel walls with a consecutive drop ofT cell numbers in the circulating blood as observed. The massivesimultaneous attachment of T cells to the blood vessel walls can causean increase in endothelial permeability and endothelial cell activation.The consequences of increased endothelial permeability are fluid shiftsfrom the intravascular compartment into interstitial tissue compartmentsincluding the CNS interstitium. Endothelial cell activation by attachedT cells can have procoagulatory effects (Monaco et al. J Leukoc Biol 71(2002) 659-668) with possible disturbances in blood flow (includingcerebral blood flow) particularly with regard to capillarymicrocirculation. Thus, CNS adverse events related to T cellredistribution in patients essentially without circulating target cellscan be the consequence of capillary leak and/or disturbances incapillary microcirculation through adherence of T cells to endothelialcells. The endothelial stress caused by one episode of T cellredistribution is tolerated by the majority of patients, while theenhanced endothelial stress caused by repeated T cell redistributionfrequently causes CNS adverse events. More than one episode of T cellredistribution may be less risky only in patients who have low baselinecounts of circulating T cells. However, also the limited endothelialstress caused by one episode of T cell redistribution can cause CNSadverse events in rare cases of increased susceptibility for such eventsas observed in 1 out of 21 patients in the bolus infusion trials withthe CD19×CD3 binding molecule.

Without being bound by theory, the transient increase of T celladhesiveness to the endothelial cells in patients who have essentiallyno circulating target cells can be explained as T cell reaction to themonovalent interaction of a conventional CD3 binding molecule, like theCD19×CD3 binding molecule (e.g. WO 99/54440), to its context dependentepitope on CD3 epsilon resulting in an allosteric change in theconformation of CD3 followed by the recruitment of Nck2 to thecytoplasmic domain of CD3 epsilon as described above. As Nck2 isdirectly linked to integrins via PINCH and ILK (FIG. 28), recruitment ofNck2 to the cytoplasmic domain of CD3 epsilon following an allostericchange in the conformation of CD3 through binding of a conventional CD3binding molecule, like the CD19×CD3 binding molecule, to its contextdependent epitope on CD3 epsilon, can increase the adhesiveness of Tcells to endothelial cells by transiently switching integrins on the Tcell surface into their more adhesive isoform via inside-out-signalling.

TABLE 3 Patient demographics and clinical outcome Best Response* Disease(CR Duration Age/ (Ann Arbor Dose Level Clearance of in Months or CohortPatient Sex Classification) [mg/m²/Day] Bone Marrow Weeks) 1 1 71/m IC,Binet C 0.0005 None SD 2 67/f MCL, Stage 0.0005 n.d. PD IV/A/E 3 67/mCLL, Stage 0.0005 n.d. MR IV/B/E 2 4 69/m MCL, Stage 0.0015 n.i. SD IV/B5 49/m MCL, Stage 0.0015 n.d. SD IV/A/S 6 71/m MCL, Stage 0.0015 n.i. PDIV/B/E 7 77/m MCL, Stage 0.0015 n.i. SD IV/B/E/S 8 65/m CLL, Stage0.0015 n.d. PD IV/B/E/S 9 75/m FL, Stage II/B 0.0015 n.i. SD 3 10 58/mMCL, Stage 0.005 n.i. PD III/B/S 11 68/f FL, Stage IV/B 0.005 n.d. SD 1265/m MCL, Stage 0.005 n.i. SD III/A/E  4^(a) 13 60/m SLL, Stage 0.015Complete PR IV/B/S 14 73/m MCL, Stage 0.015 n.i. SD II/A/E 15 44/m FL,Stage 0.015 Partial PR IV/B/E/S 16 61/m FL, Stage 0.015 Complete CR (7mo) IV/A/S 17 67/m MZL, Stage 0.015 n.i. n.e. IV/B/S 18 64/m FL, Stage0.015 n.i. PD IV/A/E 19 75/m MCL, Stage 0.015 n.i. n.e. III/A 20 65/fFL; Stage III/A 0.015 n.i. SD 21 60/m MCL, Stage 0.015 None SD IV/A/E 2267/f FL, Stage IV/B 0.015 Complete MR 23 67/m DLBCL, Stage 0.015 n.i.n.e. III/B 24 65/f FL, Stage III/A 0.015 n.d. SD 25 74/f WD, Stage IV/B0.015 Partial SD 5 26 67/m MCL, Stage 0.03 Complete SD IV/A 27 48/m FL,Stage III/A 0.03 n.i. PD 28 58/m MCL, Stage 0.03 n.i. CR (10 mo+) III/A29 45/f MCL, Stage 0.03 Partial PD IV/B 30 59/m MZL, Stage 0.03 n.i.n.e. III/A 31 43/m FL, Stage III/A 0.03 n.i. MR 6 32 72/m MCL, Stage0.06 Complete PR IV/A 33 55/m MCL, Stage 0.06 Complete CR (4 mo+) IV/B34 52/m FL, Stage IV/A 0.06 n.i. CR ^(b) (1 w+) *Centrally confirmedcomplete (CR) and partial (PR) responses by Cheson criteria in bold; MR,minimal response (≧25 to <50%); SD, stable disease; PD, progressivedisease; duration from first documentation of response in parentheses; +denotes an ongoing response ^(a)Cohort 4 was expanded to study threedifferent schedules of treatment initiation ^(b)PR after 8 weeks oftreatment that turned into a CR after an additional treatment cycle of 4weeks at the same dose following 7 weeks of treatment free intervaln.e.: not evaluable, because of treatment period <7 d n.d.: notdetermined (infiltrated, but no second biopsy performed at end oftreatment) n.i.: not infiltrated at start of treatment

TABLE 4 Incidence of adverse events observed during treatment Adverseevents regardless of relationship, occuring in ≧3 patients Grade 1-4Grade 3-4 (N = 34) N (%) N (%) Pyrexia 22 (64.7) 2 (5.9) Leukopenia 21(61.8) 11 (32.4) Lymphopenia 21 (61.8) 21 (61.8) Coagulopathy (increasein D-dimers) 16 (47.1)  6 (17.6) Enzyme abnormality (AP, LDH, CRP) 16(47.1) 10 (29.4) Hepatic function abnormality (ALT, AST, GGT) 16 (47.1)1 (2.9) Anaemia 13 (38.2)  5 (14.7) Chills 13 (38.2) 0 (0.0) Headache 12(35.3) 1 (2.9) Hypokalaemia 12 (35.3) 2 (5.9) Thrombocytopenia 12 (35.3) 6 (17.6) Weight increased 12 (35.3) 0 (0.0) Hyperglycaemia 11 (32.4) 2(5.9) Neutropenia 11 (32.4)  8 (23.5) Haematuria 10 (29.4) 0 (0.0)Oedema peripheral 10 (29.4) 2 (5.9) Anorexia  9 (26.5) 1 (2.9) Diarrhoea 9 (26.5) 0 (0.0) Weight decreased  9 (26.5) 0 (0.0) Fatigue  8 (23.5) 1(2.9) Proteinuria  8 (23.5) 0 (0.0) Hypocalcaemia  7 (20.6) 2 (5.9)Pancreatic enzyme abnormality  7 (20.6) 0 (0.0) Cough  6 (17.6) 0 (0.0)Dyspnoea  6 (17.6) 0 (0.0) Back pain  5 (14.7) 0 (0.0) Catheter sitepain  5 (14.7) 0 (0.0) Hyperbilirubinaemia  5 (14.7) 2 (5.9)Hypoalbuminaemia  5 (14.7) 0 (0.0) Hypogammaglobulinaemia  5 (14.7) 1(2.9) Hypoproteinaemia  5 (14.7) 0 (0.0) Pleural effusion  5 (14.7) 1(2.9) Vomiting  5 (14.7) 0 (0.0) Asthenia  4 (11.8) 1 (2.9) Confusionalstate  4 (11.8) 0 (0.0) Constipation  4 (11.8) 0 (0.0) Dizziness  4(11.8) 0 (0.0) Hypertension  4 (11.8) 0 (0.0) Hyponatraemia  4 (11.8) 2(5.9) Mucosal dryness  4 (11.8) 0 (0.0) Muscle spasms  4 (11.8) 0 (0.0)Nausea  4 (11.8) 0 (0.0) Night sweats  4 (11.8) 0 (0.0) Abdominal pain 3(8.8) 1 (2.9) Ascites 3 (8.8) 0 (0.0) Hypercoagulation 3 (8.8) 0 (0.0)Hyperhidrosis 3 (8.8) 0 (0.0) Hypoglobulinaemia 3 (8.8) 0 (0.0) Insomnia3 (8.8) 0 (0.0) Liver disorder 3 (8.8) 1 (2.9) Nasopharyngitis 3 (8.8) 0(0.0) Pruritus 3 (8.8) 0 (0.0) Abbreviations used are: AE, adverseevent; AP, alkaline phosphatase; LDH, lactate dehydrogenase; CRP,C-reactive protein; ALT, alanine transaminase; AST, aspartatetransaminase; GGT, gamma-glutamyl transferase; AE data from theadditional treatment cycle of patient 34 not yet included. As explainedabove, conventional CD3 binding molecules (e.g. disclosed in WO99/54440) capable of binding to a context-dependent epitope, thoughfunctional, lead to the undesired effect of T cell redistribution inpatients causing CNS adverse events. In contrast, binding molecules ofthe present invention, by binding to the context-independent N-terminal1-27 amino acids of the CD3 epsilon chain, do not lead to such T cellredistribution effects. As a consequence, the CD3 binding molecules ofthe invention are associated with a better safety profile compared toconventional CD3 binding molecules.

14. Bispecific CD3 Binding Molecules of the Invention Inducing T CellMediated Target Cell Lysis by Recognizing a Surface Target AntigenDeplete Target Antigen Positive Cells In Vivo

A Bispecific CD3 Binding Molecule of the Invention Recognizing CD33 asTarget Antigen Depletes CD33-Positive Circulating Monocytes from thePeripheral Blood of Cynomolgus Monkeys

CD33-AF5 VH-VL×I2C VH-VL (amino acid sequence: SEQ ID NO.267) wasproduced by expression in CHO cells using the coding nucleotide sequenceSEQ ID NO. 268. The coding sequences of (i) an N-terminal immunoglobulinheavy chain leader comprising a start codon embedded within a Kozakconsensus sequence and (ii) a C-terminal His₆-tag followed by a stopcodon were both attached in frame to the nucleotide sequence SEQ ID NO268 prior to insertion of the resulting DNA-fragment as obtained by genesynthesis into the multiple cloning site of the expression vectorpEF-DHFR (Raum et al. Cancer Immunol Immunother 50 (2001) 141-150).Stable transfection of DHFR-deficient CHO cells, selection forDHFR-positive transfectants secreting the CD3 binding molecule CD33-AF5VH-VL×I2C VH-VL into the culture supernatant and gene amplification withmethotrexat for increasing expression levels were carried out asdescribed (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025).Test material for treatment of cynomolgus monkeys was produced in a20-liter fermenter. Protein purification from the harvest of 3production runs was based on IMAC affinity chromatography targeting theC-terminal His6-tag of CD33-AF5 VH-VL×I2C VH-VL followed by preparativesize exclusion chromatography (SEC). The total yield of finalendotoxin-free test material was 60 mg. The analytical SEC-profile ofCD33-AF5 VH-VL×I2C VH-VL for use in cynomolgus monkeys revealed that thetest material almost exclusively consisted of monomer. The potency ofthe test material was measured in a cytotoxicity assay as described inexample 16.5 using CHO cells transfected with cynomolgus CD33 as targetcells and the macaque T cell line 4119LnPx as source of effector cells(FIG. 25). The concentration of CD33-AF5 VH-VL×I2C VH-VL required forhalf-maximal target cell lysis by the effector T cells (EC50) wasdetermined to be 2.7 ng/ml.

Young (approx. 3 years old) adult cynomolgus monkeys (Macacafascicularis) were treated by continuous intravenous infusion of CD3binding molecule CD33-AF5 VH-VL×I2C VH-VL at different flow-rates (i.e.dose levels) to study depletion of circulating CD33-positive monocytesfrom the peripheral blood. This situation is equivalent to the treatmentwith the conventional CD3 binding molecule CD19×CD3 (specific for CD19on B cells and CD3 on T cells) of those B-NHL patients, who havecirculating CD19-positive target B cells (see e.g. WO99/54440).Depletion of circulating CD19-positive target B cells from theperipheral blood had turned out as a valid surrogate for the generalclinical efficacy of the conventional CD3 binding molecule (CD19×CD3 asprovided in WO99/54440) in patients with CD19-positive B-cell malignomaslike B-NHL. Likewise, depletion of circulating CD33-positive monocytesfrom the peripheral blood is regarded as a valid surrogate of thegeneral clinical efficacy of CD33-directed bispecific CD3 bindingmolecules of the invention like CD33-AF5 VH-VL×I2C VH-VL in patientswith CD33-positive myeloid malignomas like AML (acute myeloid leukemia).

Continuous infusion was carried out according to the Swivel method asfollows: The monkeys are catheterized via the vena femoralis into thevena cava caudalis using a vein catheter. The catheter is tunneledsubcutaneously to the dorsal shoulder region and exteriorized at thecaudal scapula. Then a tube is passed through a jacket and a protectionspring. The jacket is fastened around the animal and the catheter, viathe tube, is connected to an infusion pump.

Administration solution (1.25 M lysine, 0.1% tween 80, pH 7) withouttest material was infused continuously at 48 ml/24 h for 7 days prior totreatment start to allow acclimatization of the animals to the infusionconditions. Treatment was started by adding CD33-AF5 VH-VL×I2C VH-VLtest material to the administration solution at the amount required foreach individual dose level to be tested (i.e. flow rate of CD33-AF5VH-VL×I2C VH-VL). The infusion reservoir was changed every daythroughout the whole acclimatization and treatment phase. Plannedtreatment duration was 7 days except for the 120 μg/m²/24 h dose level,where animals received 14 days of treatment.

Time courses of absolute counts in circulating T cells and CD33-positivemonocytes were determined by 4- or 3-colour FACS analysis, respectively:

Collection of Blood Samples and Routine Analysis

Blood samples (1 ml) were obtained before and 0.75, 2, 6, 12, 24, 30,48, 72 hours after start of continuous infusion with MCSP-G4 VH-VL×I2CVH-VL as well as after 7 and 14 days (and after 9 days at the 120μg/m²/24 h dose level) of treatment using EDTA-containing Vacutainer™tubes (Becton Dickinson) which were shipped for analysis at 4° C. Insome cases slight variations of these time points occurred foroperational reasons. FACS analysis of lymphocyte subpopulations wasperformed within 24-48 h after blood sample collection. Absolute numbersof leukocyte subpopulations in the blood samples were determined throughdifferential blood analysis in a routine veterinary lab.

Isolation of PBMC from Blood Samples

PBMC (peripheral blood mononuclear cells) isolation was performed by anadapted Ficoll™ gradient separation protocol. Blood was transferred atroom temperature into 5 ml Falcon™ tubes pre-loaded with 1 ml Biocoll™solution (Biochrom). Centrifugation was carried out in a swing-out rotorfor 15 min at 1700×g and 22° C. without deceleration. The PBMC above theBiocoll™ layer were isolated, washed once with FACS buffer (PBS/2% FBS[Foetal Bovine Serum; Biochrom]), centrifuged and resuspended in FACSbuffer. Centrifugation during all wash steps was carried out in aswing-out rotor for 4 min at 800×g and 4° C. If necessary, lysis oferythrocytes was performed by incubating the isolated PBMC in 3 mlerythrocyte lysis buffer (8.29 g NH₄Cl, 1.00 g KHCO₃, 0.037 g EDTA, ad1.0 l H₂O_(bidest), pH 7.5) for 5 min at room temperature followed by awashing step with FACS buffer.

Staining of PBMC with Fluorescence-Labeled Antibodies Against CellSurface Molecules

Monoclonal antibodies reactive with cynomolgus antigens were obtainedfrom Becton Dickinson (¹Cat. No. 345784, ²Cat. No. 556647, ³Cat. No.552851, ⁶Cat. No. 557710), Beckman Coulter (⁴Cat. No. IM2470) andMiltenyi (⁵Cat. No. 130-091-732) and used according to themanufacturers' recommendations. 5×10⁵-1×10⁶ cells were stained with thefollowing antibody combinations: anti-CD14¹ (FITC)×anti-CD56²(PE)×anti-CD3³ (PerCP)×anti-CD19⁴ (APC) and anti-CD14¹ (FITC)×anti-CD33⁵(PE)×anti-CD16⁶ (Alexa Fluor 647™). Cells were pelleted in V-shaped 96well multititer plates (Greiner) and the supernatant was removed. Cellpellets were resuspended in a total volume of 100 μl containing thespecific antibodies diluted in FACS buffer. Incubation was carried outin the dark for 30 min at 4° C. Subsequently, samples were washed twicewith FACS buffer and cell pellets were resuspended in FACS buffer forflowcytometric analysis.

Flowcytometric Detection of Stained Lymphocytes by FACS

Data collection was performed with a 4 color BD FACSCalibur™ (BectonDickinson). For each measurement 1×10⁴ cells of defined lymphocytesubpopulations were acquired. Statistical analysis was performed withthe program CellQuest Pro™ (Becton Dickinson) to obtain lymphocytesubpopulation percentages and to classify cell surface moleculeexpression intensity. Subsequently, percentages of single lymphocytesubsets related to total lymphocytes (i.e. B plus T plus NK cellsexcluding myeloid cells via CD14-staining) as determined by FACS werecorrelated with the lymphocyte count from the differential bloodanalysis to calculate absolute cell numbers of T cells (CD3⁺, CD56⁻,CD14⁻). Absolute numbers of CD33-positive monocytes were calculated bymultiplying the monocyte counts from the differential blood analysiswith the corresponding ratios of CD33-positive monocytes (CD33⁺, CD14⁺)to all monocytes (CD14⁺) as determined by FACS.

The percentage compared to baseline (i.e. 100%) of absolute circulatingCD33-positive monocyte counts at the end of treatment with CD33-AF5VH-VL×I2C VH-VL in 4 cohorts of 2 cynomolgus monkeys with inter-cohortdose escalation from 30 over 60 and 240 to 1000 μg/m²/24 h are shown inFIG. 26 A.

As shown in FIG. 26 A, continuous intravenous infusion of CD33-AF5VH-VL×I2C VH-VL induces depletion of circulating CD33-positive monocytesin a dose-dependent manner. While there was still no detectabledepletion of circulating CD33-positive monocytes at 30 μg/m²/24 h, afirst trend towards a reduction of CD33-positive monocyte counts becamevisible at 60 μg/m²/24 h after 7 days of treatment. At 240 μg/m²/24 hcirculating CD33-positive monocytes were almost completely depleted fromthe peripheral blood after 3 days of treatment. This was reached evenfaster at 1000 μg/m²/24 h, where depletion of the circulatingCD33-positive monocytes from the peripheral blood was completed alreadyafter 1 day of treatment. This finding was confirmed by the resultsshown in FIG. 26 B demonstrating depletion of circulating CD33-positivemonocytes by two thirds and 50% compared to the respective baseline intwo cynomolgus monkeys treated by continuous infusion with CD33-AF5VH-VL×I2C VH-VL at 120 μg/m²/24 h for 14 days.

This outcome is a clear signal clinical efficacy of the CD3 bindingmolecules of the invention in general and of bispecific CD33-directedCD3 binding molecules of the invention for the treatment ofCD33-positive malignomas like AML in particularly. Moreover, the T cellredistribution during the starting phase of treatment with CD33-AF5VH-VL×I2C VH-VL in the presence of circulating target cells (i.e.CD33-positive monocytes) seems to be less pronounced than T cellredistribution during the starting phase of treatment with conventionalCD19×CD3 constructs, as described in WO99/54440 in B-NHL patients with asignificant number of circulating target cells (i.e. CD19-positive Bcells) as shown in FIG. 22. While T cells disappear completely from thecirculation upon start of CD19×CD3 infusion and do not reappear untilthe circulating CD19-positive target B cells are depleted from theperipheral blood (FIG. 22), initial disappearance of circulating T cellsis incomplete upon infusion start with CD33-AF5 VH-VL×I2C VH-VL and Tcell counts recover still during the presence of circulatingCD33-positive target cells (FIG. 26 B). This confirms that CD3 bindingmolecules of the invention (directed against and generated against anepitope of human and non-chimpanzee primates CD3ε (epsilon) chain andbeing a part or fragment or the full length of the amino acid sequenceas provided in SEQ ID Nos. 2, 4, 6, or 8) by recognizing acontext-independent CD3 epitope show a more favorable T cellredistribution profile than conventional CD3 binding moleculesrecognizing a context-dependent CD3 epitope, like the binding moleculesprovided in WO99/54440.

15. CD3 Binding Molecules of the Invention Directed at EssentiallyContext Independent CD3 Epitopes by Inducing Less Redistribution ofCirculating T Cells in the Absence of Circulating Target Cells Reducethe Risk of Adverse Events Related to the Initiation of Treatment

Reduced T Cell Redistribution in Cynomolgus Monkeys Following Initiationof Treatment with a Representative Cross-Species Specific CD3 BindingMolecule of the Invention

MCSP-G4 VH-VL×I2C VH-VL (amino acid sequence: SEQ ID NO. 193) wasproduced by expression in CHO cells using the coding nucleotide sequenceSEQ ID NO. 194. The coding sequences of (i) an N-terminal immunoglobulinheavy chain leader comprising a start codon embedded within a Kozakconsensus sequence and (ii) a C-terminal His6-tag followed by a stopcodon were both attached in frame to the nucleotide sequence SEQ ID NO.194 prior to insertion of the resulting DNA-fragment as obtained by genesynthesis into the multiple cloning site of the expression vectorpEF-DHFR (Raum et al. Cancer Immunol Immunother 50 (2001) 141-150).Stable transfection of DHFR-deficient CHO cells, selection forDHFR-positive transfectants secreting the CD3 binding molecule MCSP-G4VH-VL×I2C VH-VL into the culture supernatant and gene amplification withmethotrexat for increasing expression levels were carried out asdescribed (Mack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025).Test material for treatment of cynomolgus monkeys was produced in a200-liter fermenter. Protein purification from the harvest was based onIMAC affinity chromatography targeting the C-terminal His6-tag ofMCSP-G4 VH-VL×I2C VH-VL followed by preparative size exclusionchromatography (SEC). The total yield of final endotoxin-free testmaterial was 40 mg. The test material consisted of 70% monomer, 30%dimer and a small contamination of higher multimer. The potency of thetest material was measured in a cytotoxicity assay as described inexample 11 using CHO cells transfected with cynomolgus MCSP as targetcells and the macaque T cell line 4119LnPx as source of effector cells(FIG. 27). The concentration of MCSP-G4 VH-VL×I2C VH-VL required forhalf-maximal target cell lysis by the effector T cells (EC50) wasdetermined to be 1.9 ng/ml.

Young (approx. 3 years old) adult cynomolgus monkeys (Macacafascicularis) were treated by continuous intravenous infusion of CD3binding molecule MCSP-G4 VH-VL×I2C VH-VL at different flow-rates (i.e.dose levels) to study redistribution of circulating T cells followinginitiation of treatment in the absence of circulating target cells.Although the CD3 binding molecule MCSP-G4 VH-VL×I2C VH-VL can recognizeboth cynomolgus MCSP and cynomolgus CD3, there are no circulating bloodcells expressing MCSP. Therefore, the only interaction possible in thecirculating blood is binding of the CD3-specific arm of MCSP-G4VH-VL×I2C VH-VL to CD3 on T cells. This situation is equivalent to thetreatment with the conventional CD3 binding molecule (CD19×CD3 bindingmolecule specific for CD19 on B cells and CD3 on T cells) of those B-NHLpatients, who have no circulating CD19-positive target B cells asdescribed in example 13.

Continuous infusion was carried out according to the Swivel method asfollows: The monkeys are catheterized via the vena femoralis into thevena cava caudalis using a vein catheter. The catheter is tunneledsubcutaneously to the dorsal shoulder region and exteriorized at thecaudal scapula. Then a tube is passed through a jacket and a protectionspring. The jacket is fastened around the animal and the catheter, viathe tube, is connected to an infusion pump.

Administration solution (1.25 M lysine, 0.1% tween 80, pH 7) withouttest material was infused continuously at 48 ml/24 h for 7 days prior totreatment start to allow acclimatization of the animals to the infusionconditions. Treatment was started by adding MCSP-G4 VH-VL×I2C VH-VL testmaterial to the administration solution at the amount required for eachindividual dose level to be tested (i.e. flow rate of MCSP-G4 VH-VL×I2CVH-VL). The infusion reservoir was changed every day throughout thewhole acclimatization and treatment phase. Treatment duration was 7days.

Time courses of absolute T-cell counts in peripheral blood weredetermined by four color FACS analysis as follows:

Collection of Blood Samples and Routine Analysis

Blood samples (1 ml) were obtained before and 0.75, 2, 6, 12, 24, 30,48, 72 hours after start of continuous infusion with MCSP-G4 VH-VL×I2CVH-VL as well as after 7 days of treatment using EDTA-containingVacutainer™ tubes (Becton Dickinson) which were shipped for analysis at4° C. In some cases slight variations of these time points occurred foroperational reasons. FACS analysis of lymphocyte subpopulations wasperformed within 24-48 h after blood sample collection. Absolute numbersof leukocyte subpopulations in the blood samples were determined throughdifferential blood analysis in a routine veterinary lab.

Isolation of PBMC from Blood Samples

PBMC (peripheral blood mononuclear cells) isolation was performed by anadapted Ficoll™ gradient separation protocol. Blood was transferred atroom temperature into 5 ml Falcon™ tubes pre-loaded with 1 ml Biocoll™solution (Biochrom). Centrifugation was carried out in a swing-out rotorfor 15 min at 1700×g and 22° C. without deceleration. The PBMC above theBiocoll™ layer were isolated, washed once with FACS buffer (PBS/2% FBS[Foetal Bovine Serum; Biochrom]), centrifuged and resuspended in FACSbuffer. Centrifugation during all wash steps was carried out in aswing-out rotor for 4 min at 800×g and 4° C. If necessary, lysis oferythrocytes was performed by incubating the isolated PBMC in 3 mlerythrocyte lysis buffer (8.29 g NH₄Cl, 1.00 g KHCO₃, 0.037 g EDTA, ad1.0 l H₂O_(bidest), pH 7.5) for 5 min at room temperature followed by awashing step with FACS buffer.

Staining of PBMC with Fluorescence-Labeled Antibodies Against CellSurface Molecules

Monoclonal antibodies reactive with cynomolgus antigens were obtainedfrom Becton Dickinson (¹Cat. No. 345784, ²Cat. No. 556647, ³Cat. No.552851) and Beckman Coulter (⁴Cat. No. IM2470) used according to themanufacturers' recommendations. 5×10⁵-1×10⁶ cells were stained with thefollowing antibody combination: anti-CD14¹ (FITC)×anti-CD56²(PE)×anti-CD3³ (PerCP)×anti-CD19⁴ (APC). Cells were pelleted in V-shaped96 well multititer plates (Greiner) and the supernatant was removed.Cell pellets were resuspended in a total volume of 100 μl containing thespecific antibodies diluted in FACS buffer. Incubation was carried outin the dark for 30 min at 4° C. Subsequently, samples were washed twicewith FACS buffer and cell pellets were resuspended in FACS buffer forflowcytometric analysis.

Flowcytometric Detection of Stained Lymphocytes by FACS

Data collection was performed with a 4 color BD FACSCalibur™ (BectonDickinson). For each measurement 1×10⁴ cells of defined lymphocytesubpopulations were acquired. Statistical analysis was performed withthe program CellQuest Pro™ (Becton Dickinson) to obtain lymphocytesubpopulation percentages and to classify cell surface moleculeexpression intensity. Subsequently, percentages of single lymphocytesubsets related to total lymphocytes (i.e. B plus T plus NK cellsexcluding myeloid cells via CD14-staining) as determined by FACS werecorrelated with the lymphocyte count from the differential bloodanalysis to calculate absolute cell numbers of T cells (CD3⁺, CD56⁻,CD14⁻).

T cell redistribution during the starting phase of treatment withMCSP-G4 VH-VL×I2C VH-VL in cynomolgus monkeys at dose levels of 60, 240and 1000 μg/m²/24 h is shown in FIG. 28. These animals showed no signsat all of any T cell redistribution during the starting phase oftreatment, i.e. T cell counts rather increased than decreased upontreatment initiation. Given that T cell redistribution is consistentlyobserved in 100% of all patients without circulating target cells, upontreatment initiation with the conventional CD3 binding molecule (e.g.CD19×CD3 construct as described in WO 99/54440) against a contextdependent CD3 epitope, it was demonstrated that substantially less Tcell redistribution in the absence of circulating target cells upontreatment initiation can be observed with a CD3 binding molecule of theinvention directed and generated against an epitope of human annon-chimpanzee primate CD3 epsilon chain as defined by the amino acidsequence of anyone of SEQ ID NOs: 2, 4, 6, or 8 or a fragment thereof.This is in clear contrast to CD3-binding molecules directed against acontext-dependent CD3 epitope, like the constructs described in WO99/54440, The binding molecules against context-independent CD3epitopes, as (inter alia) provided in any one of SEQ ID NOs: 2, 4, 6, or8 (or fragments of these sequences) provide for this substantially less(detrimental and non-desired) T cell redistribution. Because T cellredistribution during the starting phase of treatment with CD3 bindingmolecules is a major risk factor for CNS adverse events, the CD3 bindingmolecules provided herein and capable of recognizing a contextindependent CD3 epitope have a substantial advantage over the CD3binding molecules known in the art and directed againstcontext-dependent CD3 epitopes. Indeed none of the cynomolgus monkeystreated with MCSP-G4 VH-VL×I2C VH-VL showed any signs of CNS symptoms.

The context-independence of the CD3 epitope is provided in thisinvention and corresponds to the first 27 N-terminal amino acids of CD3epsilon) or fragments of this 27 amino acid stretch. Thiscontext-independent epitope is taken out of its native environmentwithin the CD3 complex and fused to heterologous amino acid sequenceswithout loss of its structural integrity. Anti-CD3 binding molecules asprovided herein and generated (and directed) against acontext-independent CD3 epitope provide for a surprising clinicalimprovement with regard to T cell redistribution and, thus, a morefavorable safety profile. Without being bound by theory, since their CD3epitope is context-independent, forming an autonomous selfsufficientsubdomain without much influence on the rest of the CD3 complex, the CD3binding molecules provided herein induce less allosteric changes in CD3conformation than the conventional CD3 binding molecules (like moleculesprovided in WO 99/54440), which recognize context-dependent CD3 epitopeslike molecules provided in WO 99/54440. As a consequence (again withoutbeing bound by theory), the induction of intracellular NcK2 recruitmentby the CD3 binding molecules provided herein is also reduced resultingin less isoform switch of T cell integrins and less adhesion of T cellsto endothelial cells. It is preferred that preparations of CD3 bindingmolecules of the invention (directed against and generated against acontext-independent epitope as defined herein) essentially consists ofmonomeric molecules. These monomeric molecules are even more efficient(than dimeric or multimeric molecules) in avoiding T cell redistributionand thus the risk of CNS adverse events during the starting phase oftreatment.

16. Generation and Characterization of CD33 and CD3 Cross-SpeciesSpecific Bispecific Single Chain Molecules 16.1. Generation of CHO CellsExpressing Human CD33

The coding sequence of human CD33 as published in GenBank (Accessionnumber NM_(—)001772) was obtained by gene synthesis according tostandard protocols. The gene synthesis fragment was designed as tocontain first a Kozak site for eukaryotic expression of the construct,followed by a 19 amino acid immunoglobulin leader peptide, followed inframe by the coding sequence of the mature human CD33 protein, followedin frame by the coding sequence of serine glycine dipeptide, ahistidine₆-tag and a stop codon (the cDNA and amino acid sequence of theconstruct is listed under SEQ ID Nos 305 and 306). The gene synthesisfragment was also designed as to introduce restriction sites at thebeginning and at the end of the fragment. The introduced restrictionsites, EcoRI at the 5′ end and SalI at the 3′ end, were utilised in thefollowing cloning procedures. The gene synthesis fragment was cloned viaEcoRI and SalI into a plasmid designated pEF-DHFR (pEF-DHFR is describedin Raum et al. Cancer Immunol Immunother 50 (2001) 141-150) followingstandard protocols. The aforementioned procedures were carried outaccording to standard protocols (Sambrook, Molecular Cloning; ALaboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press,Cold Spring Harbour, N.Y. (2001)). A clone with sequence-verifiednucleotide sequence was transfected into DHFR deficient CHO cells foreukaryotic expression of the construct. Eukaryotic protein expression inDHFR deficient CHO cells was performed as described by Kaufmann R. J.(1990) Methods Enzymol. 185, 537-566. Gene amplification of theconstruct was induced by increasing concentrations of methothrexate(MTX) to a final concentration of up to 20 nM MTX.

16.2. Generation of CHO Cells Expressing the Extracellular Domain ofMacaque CD33

The cDNA sequence of macaque CD33 was obtained by a set of 3 PCRs oncDNA from macaque monkey bone marrow prepared according to standardprotocols. The following reaction conditions: 1 cycle at 94° C. for 3minutes followed by 35 cycles with 94° C. for 1 minute, 53° C. for 1minute and 72° C. for 2 minutes followed by a terminal cycle of 72° C.for 3 minutes and the following primers were used:

1. forward primer: (SEQ ID No. 369)5′-gaggaattcaccatgccgctgctgctactgctgcccctgctgtgg gcaggggccctggctatgg-3′reverse primer: (SEQ ID No. 370) 5′-gatttgtaactgtatttggtacttcc-3′ 2.forward primer: (SEQ ID No. 371) 5′-attccgcctccttggggatcc-3′ reverseprimer: (SEQ ID No. 372) 5′-gcataggagacattgagctggatgg-3′ 3. forwardprimer: (SEQ ID No. 373) 5′-gcaccaacctgacctgtcagg-3′ reverse primer:(SEQ ID No. 374) 5′-agtgggtcgactcactgggtcctgacctctgagtattcg-3′

Those PCRs generate three overlapping fragments, which were isolated andsequenced according to standard protocols using the PCR primers, andthereby provided a portion of the cDNA sequence of macaque CD33 from thesecond nucleotide of codon +2 to the third nucleotide of codon +340 ofthe mature protein. To generate a construct for expression of macaqueCD33 a cDNA fragment was obtained by gene synthesis according tostandard protocols (the cDNA and amino acid sequence of the construct islisted under SEQ ID Nos 307 and 308). In this construct the codingsequence of macaque CD33 from amino acid+3 to +340 of the mature CD33protein was fused into the coding sequence of human CD33 replacing thehuman coding sequence of the amino acids +3 to +340. The gene synthesisfragment was also designed as to contain a Kozak site for eukaryoticexpression of the construct and restriction sites at the beginning andthe end of the fragment containing the cDNA coding for essentially thewhole extracellular domain of macaque CD33, the macaque CD33transmembrane domain and a macaque-human chimeric intracellular CD33domain. The introduced restriction sites XbaI at the 5′ end and SalI atthe 3′ end, were utilised in the following cloning procedures. The genesynthesis fragment was then cloned via XbaI and SalI into a plasmiddesignated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer ImmunolImmunother 50 (2001) 141-150). A sequence verified clone of this plasmidwas used to transfect CHO/dhfr-cells as described above.

16.3. Generation of CD33 and CD3 Cross-Species Specific BispecificAntibody Molecules Cloning of Cross-Species Specific Binding Molecules

Generally, bispecific antibody molecules, each comprising a domain witha binding specificity cross-species specific for human andnon-chimpanzee primate CD3 epsilon as well as a domain with a bindingspecificity cross-species specific for human and non-chimpanzee primateCD33, were designed as set out in the following Table 5:

TABLE 5 Formats of anti-CD3 and anti-CD33 cross-species specificbispecific molecules SEQ ID Formats of protein constructs (nucl/prot) (N→ C) 276/275 AH11HL × H2CHL 258/257 AH3HL × H2CHL 270/269 AC8HL × H2CHL264/263 AF5HL × H2CHL 288/287 F2HL × H2CHL 300/299 E11HL × H2CHL 282/281B3HL × H2CHL 294/293 B10HL × H2CHL 278/277 AH11HL × F12QHL 260/259 AH3HL× F12QHL 272/271 AC8HL × F12QHL 266/265 AF5HL × F12QHL 290/289 F2HL ×F12QHL 302/301 E11HL × F12QHL 284/283 B3HL × F12QHL 296/295 B10HL ×F12QHL 280/279 AH11HL × I2CHL 262/261 AH3HL × I2CHL 274/273 AC8HL ×I2CHL 268/267 AF5HL × I2CHL 292/291 F2HL × I2CHL 304/303 E11HL × I2CHL286/285 B3HL × I2CHL 298/297 B10HL × I2CHL

The aforementioned constructs containing the variable light-chain (L)and variable heavy-chain (H) domains cross-species specific for humanand macaque CD33 and the CD3 specific VH and VL combinationscross-species specific for human and macaque CD3 were obtained by genesynthesis. The gene synthesis fragments were designed as to containfirst a Kozak site for eukaryotic expression of the construct, followedby a 19 amino acid immunoglobulin leader peptide, followed in frame bythe coding sequence of the respective bispecific antibody molecule,followed in frame by the coding sequence of a histidine₆-tag and a stopcodon. The gene synthesis fragment was also designed as to introducesuitable restriction sites at the beginning and at the end of thefragment. The introduced restriction sites were utilized in thefollowing cloning procedures. The gene synthesis fragment was cloned viathese restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR isdescribed in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150)following standard protocols. The aforementioned procedures were carriedout according to standard protocols (Sambrook, Molecular Cloning; ALaboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press,Cold Spring Harbour, N.Y. (2001)). A clone with sequence-verifiednucleotide sequence was transfected into DHFR deficient CHO cells foreukaryotic expression of the construct. Eukaryotic protein expression inDHFR deficient CHO cells was performed as described by Kaufmann R. J.(1990) Methods Enzymol. 185, 537-566. Gene amplification of theconstruct was induced by increasing concentrations of methothrexate(MTX) to a final concentration of up to 20 nM MTX.

Expression and Purification of the Bispecific Antibody Molecules

The bispecific antibody molecules are expressed in Chinese hamster ovarycells (CHO). Eukaryotic protein expression in DHFR deficient CHO cellswas performed as described by Kaufmann R. J. (1990) Methods Enzymol.185, 537-566. Gene amplification of the constructs was induced byaddition of increasing concentrations of MTX up to final concentrationsof 20 nM MTX. After two passages of stationary culture the cells weregrown in roller bottles with nucleoside-free HyQ PF CHO liquid soymedium (with 4.0 mM L-Glutamine with 0.1% Pluronic F-68; HyClone) for 7days before harvest. The cells were removed by centrifugation and thesupernatant containing the expressed protein was stored at −20° C.Alternatively, constructs were transiently expressed in HEK 293 cells.Transfection was performed with 293fectin reagent (Invitrogen,#12347-019) according to the manufacturer's protocol.

Äkta® Explorer System (GE Health Systems) and Unicorn® Software wereused for chromatography. Immobilized metal affinity chromatography(“IMAC”) was performed using a Fractogel EMD chelate® (Merck) which wasloaded with ZnCl₂ according to the protocol provided by themanufacturer. The column was equilibrated with buffer A (20 mM sodiumphosphate buffer pH 7.2, 0.1 M NaCl) and the cell culture supernatant(500 ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.The column was washed with buffer A to remove unbound sample. Boundprotein was eluted using a two step gradient of buffer B (20 mM sodiumphosphate buffer pH 7.2, 0.1 M NaCl, 0.5 M Imidazole) according to thefollowing:

Step 1: 20% buffer B in 6 column volumesStep 2: 100% buffer B in 6 column volumes

Eluted protein fractions from step 2 were pooled for furtherpurification. All chemicals were of research grade and purchased fromSigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration chromatography was performed on a HiLoad 16/60 Superdex200 prep grade column (GE/Amersham) equilibrated with Equi-buffer (25 mMCitrate, 200 mM Lysine, 5% Glycerol, pH 7.2). Eluted protein samples(flow rate 1 ml/min) were subjected to standard SDS-PAGE and WesternBlot for detection. Prior to purification, the column was calibrated formolecular weight determination (molecular weight marker kit, Sigma MWGF-200). Protein concentrations were determined using OD280 nm.

Purified bispecific antibody protein was analyzed in SDS PAGE underreducing conditions performed with pre-cast 4-12% Bis Tris gels(Invitrogen). Sample preparation and application were performedaccording to the protocol provided by the manufacturer. The molecularweight was determined with MultiMark protein standard (Invitrogen). Thegel was stained with colloidal Coomassie (Invitrogen protocol). Thepurity of the isolated protein is >95% as determined by SDS-PAGE.

The bispecific antibody has a molecular weight of about 52 kDa undernative conditions as determined by gel filtration in PBS. All constructswere purified according to this method.

Western Blot was performed using an Optitran® BA-S83 membrane and theInvitrogen Blot Module according to the protocol provided by themanufacturer. For detection of the bispecific antibody proteinantibodies an anti-His Tag antibody was used (Penta His, Qiagen). AGoat-anti-mouse Ig antibody labeled with alkaline phosphatase (AP)(Sigma) was used as secondary antibody and BCIP/NBT (Sigma) assubstrate. A single band was detected at 52 kD corresponding to thepurified bispecific antibody.

16.4. Flow Cytometric Binding Analysis of the CD33 and CD3 Cross-SpeciesSpecific Bispecific Antibodies

In order to test the functionality of the cross-species specificbispecific antibody constructs regarding the capability to bind to humanand macaque CD33 and CD3, respectively, a FACS analysis was performed.For this purpose CHO cells transfected with human CD33 as described inExample 16.1 and the human CD3 positive T cell leukemia cell lineHPB-ALL (DSMZ, Braunschweig, ACC483) were used to test the binding tohuman antigens. The binding reactivity to macaque antigens was tested byusing the generated macaque CD33 transfectant described in Example 16.2and macaque PBMC (preparation of macaque PBMC was performed by Ficollgradient centrifugation of peripheral blood from macaque monkeysaccording to standard protocolls). 200.000 cells of the respective celllines of PBMC were incubated for 30 min. on ice with 50 μl of thepurified protein of the cross-species specific bispecific antibodyconstructs (5 μg/ml) or cell culture supernatant of transfected cellsexpressing the cross-species specific bispecific antibody constructs.The cells were washed twice in PBS with 2% FCS and binding of theconstruct was detected with a murine anti-His antibody (Penta Hisantibody; Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS). After washing,bound anti-His antibodies were detected with an Fc gamma-specificantibody (Dianova) conjugated to phycoerythrin, diluted 1:100 in PBSwith 2% FCS. Supernatant of untransfected CHO cells was used as negativecontrol.

Flow cytometry was performed on a FACS-Calibur apparatus; the CellQuestsoftware was used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

The specific binding of human and non-chimpanzee primate CD3 of the CD3binding molecules of the invention was clearly detectable as shown inFIG. 29. In the FACS analysis all constructs show binding to CD3 andCD33 as compared to the respective negative controls. Cross-speciesspecificity of the bispecific antibodies to human and macaque CD3 andCD33 antigens is demonstrated.

16.5. Bioactivity of CD33 and CD3 Cross-Species Specific BispecificAntibodies

Bioactivity of the generated bispecific antibodies was analyzed bychromium 51 (⁵¹Cr) release in vitro cytotoxicity assays using the CD33positive cell lines described in Examples 16.1 and 16.2. As effectorcells stimulated human CD4/CD56 depleted PBMC or the macaque T cell line4119LnPx were used as specified in the respective figures.

Generation of stimulated human PBMC was performed as follows:

A Petri dish (85 mm diameter, Nunc) was coated with a commerciallyavailable anti-CD3 specific antibody (e.g. OKT3, Othoclone) in a finalconcentration of 1 μg/ml for 1 hour at 37° C. Unbound protein wasremoved by one washing step with PBS. The fresh PBMC were isolated fromperipheral blood (30-50 ml human blood) by Ficoll gradientcentrifugation according to standard protocols. 3-5×10⁷ PBMC were addedto the precoated petri dish in 50 ml of RPMI 1640 with stabilizedglutamine/10% FCS/IL-2 20 U/ml (Proleukin, Chiron) and stimulated for 2days. On the third day the cells were collected and washed once withRPMI 1640. IL-2 was added to a final concentration of 20 U/ml and thecells were cultivated again for one day in the same cell culture mediumas above.

Target cells were washed twice with PBS and labeled with 11.1 MBq ⁵¹Crin a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37° C.Subsequently the labeled target cells were washed 3 times with 5 ml RPMIand then used in the cytotoxicity assay. The assay was performed in a 96well plate in a total volume of 250 μl supplemented RPMI (as above) withan E:T ratio of 10:1. 1 μg/ml of the cross-species specific bispecificantibody molecules and 20 threefold dilutions thereof were applied. Theassay time was 18 hours and cytotoxicity was measured as relative valuesof released chromium in the supernatant related to the difference ofmaximum lysis (addition of Triton-X) and spontaneous lysis (withouteffector cells). All measurements were done in quadruplicates.Measurement of chromium activity in the supernatants was performed witha Wizard 3″ gamma counter (Perkin Elmer Life Sciences GmbH, Köln,Germany). Analysis of the experimental data was performed with Prism 4for Windows (version 4.02, GraphPad Software Inc., San Diego, Calif.,USA). Sigmoidal dose response curves typically have R² values >0.90 asdetermined by the software. EC₅₀ values calculated by the analysisprogram were used for comparison of bioactivity.

As shown in FIG. 30, all of the generated cross-species specificbispecific constructs demonstrate cytotoxic activity against human CD33positive target cells elicited by stimulated human CD4/CD56 depletedPBMC and against macaque CD33 positive target cells elicited by themacaque T cell line 4119LnPx.

17. Purification of Cross-Species Specific Bispecific Single chainMolecules by an Affinity Procedure Based on the Context Independent CD3Epsilon Epitope Corresponding to the N-Terminal Amino Acids 1-27 17.1Generation of an Affinity Column Displaying the Isolated ContextIndependent Human CD3 Epsilon Epitope Corresponding to the N-TerminalAmino Acids 1-27

The plasmid for expression of the construct 1-27 CD3-Fc consisting ofthe 1-27 N-terminal amino acids of the human CD3 epsilon chain fused tothe hinge and Fc gamma region of human immunoglobulin IgG1 describedabove (Example 3; cDNA sequence and amino acid sequence of therecombinant fusion protein are listed under SEQ ID NOs 230 and 229) wastransfected into DHFR deficient CHO cells for eukaryotic expression ofthe construct. Eukaryotic protein expression in DHFR deficient CHO cellswas performed as described by Kaufmann R. J. (1990) Methods Enzymol.185, 537-566. Gene amplification of the construct was induced byincreasing concentrations of methotrexate (MTX) to a final concentrationof up to 20 nM MTX. After two passages of stationary culture the cellswere grown in roller bottles with nucleoside-free HyQ PF CHO liquid soymedium (with 4.0 mM L-Glutamine with 0.1% Pluronic F-68; HyClone) for 7days before harvest. The cells were removed by centrifugation and thesupernatant containing the expressed protein was stored at −20° C. Forthe isolation of the fusion protein a goat anti-human fc affinity columnwas prepared according to standard protocols using a commerciallyavailable affinity purified goat anti-human IgG fc fragment specificantibody with minimal cross-reaction to bovine, horse, and mouse serumproteins (Jackson ImmunoResearch Europe Ltd.). Using this affinitycolumn the fusion protein was isolated out of cell culture supernatanton an Äkta Explorer System (GE Amersham) and eluted by citric acid. Theeluate was neutralized and concentrated. After dialysis against aminefree coupling buffer the purified fusion protein was coupled to anN-Hydroxy-Succinimide NHS activated 1 ml HiTrap column (GE Amersham).

After coupling remaining NHS groups were blocked and the column waswashed and stored at 5° C. in storage buffer containing 0.1% sodiumazide.

17.2 Purification of Cross-Species Specific Bispecific Single ChainMolecules Using a Human CD3 Peptide Affinity Column

200 ml cell culture supernatant of cells expressing cross-speciesspecific bispecific single chain molecules were 0.2 μm sterile filteredand applied to the CD3 peptide affinity column using an Äkta Explorersystem (GE Amersham).

The column was then washed with phosphate buffered saline PBS pH 7.4 towash out unbound sample. Elution was done with an acidic buffer pH 3.0containing 20 mM Citric acid and 1 M sodium chloride. Eluted protein wasneutralized immediately by 1 M Trishydroxymethylamine TRIS pH 8.3contained in the collection tubes of the fraction collector.

Protein analysis was done by SDS PAGE and Western Blot.

For SDS PAGE BisTris Gels 4-12% are used (Invitrogen). The runningbuffer was 1×MES-SDS-Puffer (Invitrogen). As protein standard 15 μlprestained Sharp Protein Standard (Invitrogen) was applied.Electrophoresis was performed for 60 minutes at 200 volts 120 mA max.Gels were washed in demineralised water and stained with Coomassie forone hour. Gels are destained in demineralised water for 3 hours.Pictures are taken with a Syngene Gel documentation system.

For Western Blot a double of the SDS PAGE gel was generated and proteinswere electroblotted onto a nitrocellulose membrane. The membrane wasblocked with 2% bovine serum albumin in PBS and incubated with abiotinylated murine Penta His antibody (Qiagen). As secondary reagent astreptavidin alkaline phosphatase conjugate (DAKO) was used. Blots weredeveloped with BCIP/NBT substrate solution (Pierce).

As demonstrated in FIGS. 31, 32 and 33 the use of a human CD3 peptideaffinity column as described above allows the highly efficientpurification of the bispecific single chain molecules from cell culturesupernatant. The cross-species specific anti-CD3 single chain antibodiescontained in the bispecific single chain molecules therefore enable viatheir specific binding properties an efficient generic one-step methodof purification for the cross-species specific bispecific single chainmolecules, without the need of any tags solely attached for purificationpurposes.

18. Generic Pharmacokinetic Assay for Cross-Species Specific BispecificSingle Chain Molecules 18.1 Production of 1-27 CD3-Fc for Use in thePharmacokinetic Assay

The coding sequence of the 1-27 N-terminal amino acids of the human CD3epsilon chain fused to the hinge and Fc gamma region of humanimmunoglobulin IgG1 was obtained by gene synthesis according to standardprotocols (cDNA sequence and amino acid sequence of the recombinantfusion protein are listed under SEQ ID NOs 309 and 310). The genesynthesis fragment was designed as to contain first a Kozak site foreukaryotic expression of the construct, followed by a 19 amino acidimmunoglobulin leader peptide, followed in frame by the coding sequenceof the first 27 amino acids of the extracellular portion of the maturehuman CD3 epsilon chain, followed in frame by the coding sequence of thehinge region and Fc gamma portion of human IgG1 and a stop codon. Thegene synthesis fragment was also designed as to introduce restrictionsites at the beginning and at the end of the cDNA coding for the fusionprotein. The introduced restriction sites, EcoRI at the 5′ end and SalIat the 3′ end, were utilised in the following cloning procedures. Thegene synthesis fragment was cloned via EcoRI and SalI into a plasmiddesignated pEF-DHFR (pEF-DHFR is described in Raum et al. Cancer ImmunolImmunother 50 (2001) 141-150) following standard protocols. Theafore-mentioned procedures were carried out according to standardprotocols (Sambrook, Molecular Cloning; A Laboratory Manual, 3rdedition, Cold Spring Harbour Laboratory Press, Cold Spring Harbour, N.Y.(2001)). A clone with sequence-verified nucleotide sequence wastransfected into DHFR deficient CHO cells for eukaryotic expression ofthe construct. Eukaryotic protein expression in DHFR deficient CHO cellswas performed as described by Kaufmann R. J. (1990) Methods Enzymol.185, 537-566. Gene amplification of the construct was induced byincreasing concentrations of methotrexate (MTX) to a final concentrationof up to 20 nM MTX. After two passages of stationary culture the cellswere grown in roller bottles with nucleoside-free HyQ PF CHO liquid soymedium (with 4.0 mM L-Glutamine with 0.1% Pluronic F-68; HyClone) for 7days before harvest. The cells were removed by centrifugation and thesupernatant containing the expressed protein was stored at −20° C. Forthe isolation of the fusion protein a goat anti-human fc affinity columnwas prepared according to standard protocols using a commerciallyavailable affinity purified goat anti-human IgG fc fragment specificantibody with minimal cross-reaction to bovine, horse, and mouse serumproteins (Jackson ImmunoResearch Europe Ltd.). Using this affinitycolumn the fusion protein was isolated out of cell culture supernatanton an Akta Explorer System (GE Amersham) and eluted by citric acid. Theeluate was neutralized and concentrated.

18.2 Pharmacokinetic Assay for Cross-Species Specific Bispecific SingleChain Molecules

The assay is based on the ECL-ELISA technology using ruthenium labelleddetection on carbon plates measured on a Sektor Imager device (MSD). Ina first step, carbon plates (MSD High Bind Plate 96 well Cat: L15xB-3)were coated with 5 μl/well at 50 ng/ml of the purified 1-27 CD3-Fcdescribed in Example 18.1. The plate was then dried overnight at 25° C.Subsequently plates were blocked with 5% BSA (Paesel&Lorei #100568) inPBS at 150 μl/well for 1 h at 25° C. in an incubator while shaking (700rpm). In the next step plates were washed three times with 0.05% Tweenin PBS. A standard curve in 50% macaque serum in PBS was generated byserial 1:4 dilution starting at 100 ng/ml of the respectivecross-species specific bispecific single chain molecule to be detectedin the assay. Quality control (QC) samples were prepared in 50% macaqueserum in PBS ranging from 1 ng/ml to 50 ng/ml of the respectivecross-species specific bispecific single chain molecule dependent on theexpected sample serum concentrations. Standard, QC or unknown sampleswere transferred to the carbon plates at 10 μl/well and incubated for 90min at 25° C. in the incubator while shaking (700 rpm). Subsequentlyplates were washed three times with 0.05% Tween in PBS. For detection 25μl/well of penta-His-Biotin antibody (Qiagen, 200 μg/ml in 0.05% Tweenin PBS) was added and incubated for 1 h at 25° C. in an incubator whileshaking (700 rpm). In a second detection step 25 μl/wellStreptavidin-SulfoTag solution (MSD; Cat: R32AD-1; Lot: WO010903) wasadded and incubated for 1 h at 25° C. in an incubator while shaking (700rpm). Subsequently plates were washed three times with 0.05% Tween inPBS. Finally 150 μl/well MSD Reading Buffer (MSD, Cat: R9ZC-1) was addedand plates were read in the Sektor Imager device.

FIGS. 34 and 35 demonstrate the feasibility of detection ofcross-species specific bispecific single chain molecules in serumsamples of macaque monkeys for cross-species specific bispecific singlechain molecules. The cross-species specific anti-CD3 single chainantibodies contained in the bispecific single chain molecules enabletherefore via their specific binding properties a highly sensitivegeneric assay for detection of the cross-species specific bispecificsingle chain molecules. The assay set out above can be used in thecontext of formal toxicological studies that are needed for drugdevelopment and can be easily adapted for measurement of patient samplesin connection with the clinical application of cross-species specificbispecific single chain molecules.

19. Generation of Recombinant Transmembrane Fusion Proteins of theN-Terminal Amino Acids 1-27 of CD3 Epsilon from Different Non-ChimpanzeePrimates Fused to EpCAM from Cynomolgus Monkey (1-27 CD3-EpCAM) 19.1Cloning and Expression of 1-27 CD3-EpCAM

CD3 epsilon was isolated from different non-chimpanzee primates(marmoset, tamarin, squirrel monkey) and swine. The coding sequences ofthe 1-27 N-terminal amino acids of CD3 epsilon chain of the maturehuman, common marmoset (Callithrix jacchus), cottontop tamarin (Saguinusoedipus), common squirrel monkey (Saimiri sciureus) and domestic swine(Sus scrofa; used as negative control) fused to the N-terminus of Flagtagged cynomolgus EpCAM were obtained by gene synthesis according tostandard protocols (cDNA sequence and amino acid sequence of therecombinant fusion proteins are listed under SEQ ID NOs 231 to 240). Thegene synthesis fragments were designed as to contain first a BsrGI siteto allow for fusion in correct reading frame with the coding sequence ofa 19 amino acid immunoglobulin leader peptide already present in thetarget expression vector, which was followed in frame by the codingsequence of the N-terminal 1-27 amino acids of the extracellular portionof the mature CD3 epsilon chains, which was followed in frame by thecoding sequence of a Flag tag and followed in frame by the codingsequence of the mature cynomolgus EpCAM transmembrane protein. The genesynthesis fragments were also designed to introduce a restriction siteat the end of the cDNA coding for the fusion protein. The introducedrestriction sites BsrGI at the 5′ end and SalI at the 3′ end, wereutilized in the following cloning procedures. The gene synthesisfragments were then cloned via BsrGI and SalI into a derivative of theplasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al. CancerImmunol Immunother 50 (2001) 141-150), which already contains the codingsequence of the 19 amino acid immunoglobulin leader peptide followingstandard protocols. Sequence verified plasmids were used to transfectDHFR deficient CHO cells for eukaryotic expression of the construct.Eukaryotic protein expression in DHFR deficient CHO cells was performedas described by Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566.Gene amplification of the construct was induced by increasingconcentrations of methotrexate (MTX) to a final concentration of up to20 nM MTX.

Transfectants were tested for cell surface expression of the recombinanttransmembrane protein via an FACS assay according to standard protocols.For that purpose a number of 2.5×10⁵ cells were incubated with 50 μl ofthe anti-Flag M2 antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen,Germany) at 5 μg/ml in PBS with 2% FCS. Bound antibody was detected withan R-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment, goatanti-mouse IgG, Fc-gamma fragment specific 1:100 in PBS with 2% FCS(Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Flowcytometry was performed on a FACS-Calibur apparatus, the CellQuestsoftware was used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

Expression of the Flag tagged recombinant transmembrane fusion proteinsconsisting of cynomolgus EpCAM and the 1-27 N-terminal amino acids ofthe human, marmoset, tamarin, squirrel monkey and swine CD3 epsilonchain respectively on transfected cells is clearly detectable (FIG. 36).

19.2 Cloning and Expression of the Cross-Species Specific Anti-CD3Single Chain Antibody I2C HL in Form of an IgG1 Antibody

In order to provide improved means of detection of binding of thecross-species specific single chain anti-CD3 antibody the I2C VHVLspecificity is converted into an IgG1 antibody with murine IgG1 andmurine kappa constant regions. cDNA sequences coding for the heavy chainof the IgG antibody were obtained by gene synthesis according tostandard protocols. The gene synthesis fragments were designed as tocontain first a Kozak site to allow for eukaryotic expression of theconstruct, which is followed by an 19 amino acid immunoglobulin leaderpeptide, which is followed in frame by the coding sequence of the heavychain variable region or light chain variable region, followed in frameby the coding sequence of the heavy chain constant region of murine IgG1as published in GenBank (Accession number AB097849) or the codingsequence of the murine kappa light chain constant region as published inGenBank (Accession number D14630), respectively.

Restriction sites were introduced at the beginning and the end of thecDNA coding for the fusion protein. Restriction sites EcoRI at the 5′end and SalI at the 3′ end were used for the following cloningprocedures. The gene synthesis fragments were cloned via EcoRI and SalIinto a plasmid designated pEF-DHFR (pEF-DHFR is described in Raum et al.Cancer Immunol Immunother 50 (2001) 141-150) for the heavy chainconstruct and pEFADA (pEFADA is described in Raum et al. Cancer ImmunolImmunother 50 (2001) 141-150) for the light chain construct according tostandard protocols. Sequence verified plasmids were used forco-transfection of respective light and heavy chain constructs into DHFRdeficient CHO cells for eukaryotic expression of the construct.Eukaryotic protein expression in DHFR deficient CHO cells was performedas described by Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566.Gene amplification of the constructs was induced by increasingconcentrations of methotrexate (MTX) to a final concentration of up to20 nM MTX and deoxycoformycin (dCF) to a final concentration of up to300 nM dCF. After two passages of stationary culture cell culturesupernatant was collected and used in the subsequent experiment.

19.3 Binding of the Cross-Species Specific Anti-CD3 Single ChainAntibody I2C HL in Form of an IgG1 Antibody to 1-27 CD3-EpCAM

Binding of the generated I2C IgG1 construct to the 1-27 N-terminal aminoacids of the human, marmoset, tamarin and squirrel monkey CD3 epsilonchains respectively fused to cynomolgus Ep-CAM as described in Example19.1 was tested in a FACS assay according to standard protocols. Forthat purpose a number of 2.5×10⁵ cells were incubated with 50 μl of cellculture supernatant containing the I2C IgG1 construct as described inExample 19.2. The binding of the antibody was detected with anR-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment, goatanti-mouse IgG, Fc-gamma fragment specific, diluted 1:100 in PBS with 2%FCS (Jackson ImmunoResearch Europe Ltd., Newmarket, Suffolk, UK). Flowcytometry was performed on a FACS-Calibur apparatus, the CellQuestsoftware was used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

As shown in FIG. 37 binding of the I2C IgG1 construct to thetransfectants expressing the recombinant transmembrane fusion proteinsconsisting of the 1-27 N-terminal amino acids of CD3 epsilon of human,marmoset, tamarin or squirrel monkey fused to cynomolgus EpCAM ascompared to the negative control consisting of the 1-27 N-terminal aminoacids of CD3 epsilon of swine fused to cynomolgus EpCAM was observed.Thus multi-primate cross-species specificity of I2C was demonstrated.Signals obtained with the anti Flag M2 antibody and the I2C IgG1construct were comparable, indicating a strong binding activity of thecross-species specific specificity I2C to the N-terminal amino acids1-27 of CD3 epsilon.

20. Binding of the Cross-Species Specific Anti-CD3 Binding Molecule I2Cto the Human CD3 Epsilon Chain with and without N-Terminal His6 Tag

A chimeric IgG1 antibody with the binding specificity I2C as describedin Example 19.2 specific for CD3 epsilon was tested for binding to humanCD3 epsilon with and without N-terminal His6 tag. Binding of theantibody to the EL4 cell lines transfected with His6-human CD3 epsilonas described in Example 6.1 and wild-type human CD3 epsilon as describedin Example 5.1 respectively was tested by a FACS assay according tostandard protocols. 2.5×10⁵ cells of the transfectants were incubatedwith 50 μl of cell culture supernatant containing the I2C IgG1 constructor 50 μl of the respective control antibodies at 5 μg/ml in PBS with 2%FCS. As negative control an appropriate isotype control and as positivecontrol for expression of the constructs the CD3 specific antibodyUCHT-1 were used respectively. Detection of the His6 tag was performedwith the penta His antibody (Qiagen). The binding of the antibodies wasdetected with a R-Phycoerythrin-conjugated affinity purified F(ab′)2fragment, goat anti-mouse IgG, Fc-gamma fragment specific, diluted 1:100in PBS with 2% FCS (Jackson ImmunoResearch Europe Ltd., Newmarket,Suffolk, UK). Flow cytometry was performed on a FACS-Calibur apparatus,the CellQuest software was used to acquire and analyze the data (BectonDickinson biosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

Comparable binding of the anti-human CD3 antibody UCHT-1 to bothtransfectants demonstrates approximately equal levels of expression ofthe constructs. The binding of the penta His antibody confirmed thepresence of the His6 tag on the His6-human CD3 construct but not on thewild-type construct.

Compared to the EL4 cell line transfected with wild-type human CD3epsilon a clear loss of binding of the I2C IgG1 construct to human-CD3epsilon with an N-terminal His 6 tag was detected. These results showthat a free N-terminus of CD3 epsilon is essential for binding of thecross-species specific anti-CD3 binding specificity I2C to the human CD3epsilon chain (FIG. 28).

21. Generation of CD33 and CD3 Cross-Species Specific Bispecific SingleChain Molecules 21.1 Generation of CD33 and CD3 Cross-Species SpecificBispecific Single Chain Molecules

Generally, bispecific single chain antibody molecules, each comprising adomain with a binding specificity cross-species specific for human andmacaque CD3epsilon as well as a domain with a binding specificitycross-species specific for human and macaque CD33, were designed as setout in the following Table 6:

TABLE 6 Formats of anti-CD3 and anti-CD33 cross-species specificbispecific single chain antibody molecules SEQ ID Formats of proteinconstructs (nucl/prot) (N → C) 316/315 I2CHL × AF5HL 314/313 F12QHL ×AF5HL 312/311 H2CHL × AF5HL

The aforementioned constructs containing the variable light-chain (L)and variable heavy-chain (H) domains cross-species specific for humanand macaque CD33 and the CD3 specific VH and VL combinationscross-species specific for human and macaque CD3 were obtained by genesynthesis. The gene synthesis fragments were designed as to containfirst a Kozak site for eukaryotic expression of the construct, followedby a 19 amino acid immunoglobulin leader peptide, followed in frame bythe coding sequence of the respective bispecific single chain antibodymolecule, followed in frame by the coding sequence of a histidine₆-tagand a stop codon. The gene synthesis fragment was also designed as tointroduce suitable restriction sites at the beginning and at the end ofthe fragment. The introduced restriction sites were utilised in thefollowing cloning procedures. The gene synthesis fragment was cloned viathese restriction sites into a plasmid designated pEF-DHFR (pEF-DHFR isdescribed in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150)following standard protocols. The aforementioned procedures were carriedout according to standard protocols (Sambrook, Molecular Cloning; ALaboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press,Cold Spring Harbour, N.Y. (2001)). A clone with sequence-verifiednucleotide sequence was transfected into DHFR deficient CHO cells foreukaryotic expression of the construct. Eukaryotic protein expression inDHFR deficient CHO cells was performed as described by Kaufmann R. J.(1990) Methods Enzymol. 185, 537-566. Gene amplification of theconstruct was induced by increasing concentrations of methotrexate (MTX)to a final concentration of up to 20 nM MTX. After two passages ofstationary culture cell culture supernatant was collected and used inthe subsequent experiments.

21.2 Flow Cytometric Binding Analysis of the CD33 and CD3 Cross-SpeciesSpecific Bispecific Antibodies

In order to test the functionality of the cross-species specificbispecific antibody constructs regarding the capability to bind to humanand macaque CD33 and CD3, respectively, a FACS analysis is performed.For this purpose CHO cells transfected with human CD33 as described inExample 16.1 and the human CD3 positive T cell leukemia cell lineHPB-ALL (DSMZ, Braunschweig, ACC483) were used to test the binding tohuman antigens. The binding reactivity to macaque antigens was tested byusing the generated macaque CD33 transfectant described in Example 16.2and macaque PBMC (preparation of macaque PBMC was performed by Ficollgradient centrifugation of peripheral blood from macaque monkeysaccording to standard protocols). 200000 cells of the respective celllines or PBMC were incubated for 30 min. on ice with 50 μl of cellculture supernatant of transfected cells expressing the cross-speciesspecific bispecific antibody constructs. The cells were washed twice inPBS with 2% FCS and binding of the construct was detected with a murineanti-His antibody (Penta His antibody; Qiagen; diluted 1:20 in 50 μl PBSwith 2% FCS). After washing, bound anti-His antibodies were detectedwith an Fc gamma-specific antibody (Dianova) conjugated tophycoerythrin, diluted 1:100 in PBS with 2% FCS. Supernatant ofuntransfected CHO cells was used as negative control.

Flow cytometry was performed on a FACS-Calibur apparatus; the CellQuestsoftware was used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

The bispecific binding of the single chain molecules listed above, whichwere cross-species specific for CD33 and cross-species specific forhuman and non-chimpanzee primate CD3 was clearly detectable as shown inFIG. 41. In the FACS analysis all constructs showed binding to CD3 andCD33 as compared to the respective negative controls. Cross-speciesspecificity of the bispecific antibodies to human and macaque CD3 andCD33 antigens was demonstrated.

21.3. Bioactivity of CD33 and CD3 Cross-Species Specific BispecificSingle Chain Antibodies

Bioactivity of the generated bispecific single chain antibodies wasanalyzed by chromium 51 (⁵¹Cr) release in vitro cytotoxicity assaysusing the CD33 positive cell lines described in Examples 16.1 and 16.2.As effector cells stimulated human CD4/CD56 depleted PBMC or the macaqueT cell line 4119LnPx were used as specified in the respective figures.

A Petri dish (145 mm diameter, Greiner bio-one GmbH, Kremsmunster) wascoated with a commercially available anti-CD3 specific antibody (e.g.OKT3, Orthoclone) in a final concentration of 1 μg/ml for 1 hour at 37°C. Unbound protein was removed by one washing step with PBS. The freshPBMC were isolated from peripheral blood (30-50 ml human blood) byFicoll gradient centrifugation according to standard protocols. 3-5×10⁷PBMC were added to the precoated Petri dish in 120 ml of RPMI 1640 withstabilized glutamine/10% FCS/IL-2 20 U/ml (Proleukin, Chiron) andstimulated for 2 days. On the third day the cells were collected andwashed once with RPMI 1640. IL-2 is added to a final concentration of 20U/ml and the cells were cultivated again for one day in the same cellculture medium as above.

By depletion of CD4+ T cells and CD56+ NK cells according to standardprotocols CD8+ cytotoxic T lymphocytes (CTLs) were enriched.

Target cells were washed twice with PBS and labelled with 11.1 MBq ⁵¹Crin a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37° C.Subsequently the labelled target cells were washed 3 times with 5 mlRPMI and then used in the cytotoxicity assay. The assay was performed ina 96 well plate in a total volume of 250 μl supplemented RPMI (as above)with an E:T ratio of 10:1. Supernatant of cells expressing thecross-species specific bispecific single chain antibody molecules in afinal concentration of 50% and 20 threefold dilutions thereof wereapplied. The assay time is 18 hours and cytotoxicity was measured asrelative values of released chromium in the supernatant related to thedifference of maximum lysis (addition of Triton-X) and spontaneous lysis(without effector cells). All measurements were done in quadruplicates.Measurement of chromium activity in the supernatants was performed witha Wizard 3″ gamma counter (Perkin Elmer Life Sciences GmbH, Köln,Germany). Analysis of the experimental data was performed with Prism 4for Windows (version 4.02, GraphPad Software Inc., San Diego, Calif.,USA). Sigmoidal dose response curves typically have R² values >0.90 asdetermined by the software. EC₅₀ values calculated by the analysisprogram were used for comparison of bioactivity.

As shown in FIG. 42, all of the generated cross-species specificbispecific single chain antibody constructs demonstrate cytotoxicactivity against human CD33 positive target cells elicited by stimulatedhuman CD4/CD56 depleted PBMC and against macaque CD33 positive targetcells elicited by the macaque T cell line 4119LnPx.

22. Redistribution of Circulating Chimpanzee T Cells Upon Exposure to aConventional Bispecific CD3 Binding Molecule Directed at a TargetMolecule which is Absent from Circulating Blood Cells

A single male chimpanzee was subjected to dose escalation withintravenous single-chain EpCAM/CD3-bispecific antibody construct(Schlereth (2005) Cancer Res 65: 2882). Like in the conventionalsingle-chain CD19/CD3-bispecific antibody construct (Loffler (2000,Blood, Volume 95, Number 6) or WO 99/54440), the CD3 arm of saidEpCAM/CD3-construct is also directed against a conventional contextdependent epitope of human and chimpanzee CD3. At day 0, the animalreceived 50 ml PBS/5% HSA without test material, followed by 50 mlPBS/5% HSA plus single-chain EpCAM/CD3-bispecific antibody construct at1.6, 2.0, 3.0 and 4.5 μg/kg on days 7, 14, 21 and 28, respectively. Theinfusion period was 2 hours per administration. For each weekly infusionthe chimpanzee was sedated with 2-3 mg/kg Telazol intramuscularly,intubated and placed on isoflurane/O₂ anesthesia with stable mean bloodpressures. A second intravenous catheter was placed in an opposite limbto collect (heparinized) whole blood samples at the time pointsindicated in FIG. 43 for FACS analysis of circulating blood cells. Afterstandard erythrocyte lysis, T cells were stained with a FITC-labeledantibody reacting with chimpanzee CD2 (Becton Dickinson) and thepercentage of T cells per total lymphocytes determined by flowcytometry.As shown in FIG. 43, every administration of single-chainEpCAM/CD3-bispecific antibody construct induced a rapid drop ofcirculating T cells as observed with single-chain CD19/CD3-bispecificantibody construct in B-NHL patients, who had essentially no circulatingtarget B (lymphoma) cells. As there are no EpCAM-positive target cellsin the circulating blood of humans and chimpanzees, the drop ofcirculating T cells upon exposure to the single-chainEpCAM/CD3-bispecific antibody construct can be attributed solely to asignal, which the T cells receive through pure interaction of the CD3arm of the construct with a conventional context dependent CD3 epitopein the absence of any target cell mediated crosslinking. Like theredistribution of T cells induced through their exposure to single-chainCD19/CD3-bispecific antibody construct in B-NHL patients, who hadessentially no circulating target B (lymphoma) cells, the T cellredistribution in the chimpanzee upon exposure to the single-chainEpCAM/CD3-bispecific antibody construct can be explained by aconformational change of CD3 following the binding event to a contextdependent CD3 epitope further resulting in the transient increase of Tcell adhesiveness to blood vessel endothelium (see Example 13). Thisfinding confirms, that conventional CD3 binding molecules directed tocontext dependent CD3 epitopes—solely through this interaction—can leadto a redistribution pattern of peripheral blood T cells, which isassociated with the risk of CNS adverse events in humans as describe inExample 13.

23. Specific Binding of scFv Clones to the N-Terminus of Human CD3Epsilon

23.1 Bacterial Expression of scFv Constructs in E. coli XL1 Blue

As previously mentioned, E. coli XL1 Blue transformed withpComb3H5Bhis/Flag containing a VL- and VH-segment produce soluble scFvin sufficient amounts after excision of the gene III fragment andinduction with 1 mM IPTG. The scFv-chain is exported into the periplasmawhere it folds into a functional conformation.

The following scFv clones were chosen for this experiment:

i) ScFvs 4-10, 3-106, 3-114, 3-148, 4-48, 3-190 and 3-271 as describedin WO 2004/106380.ii) ScFvs from the human anti-CD3epsilon binding clones H2C, F12Q andI2C as described herein.

For periplasmic preparations, bacterial cells transformed with therespective scFv containing plasmids allowing for periplasmic expressionwere grown in SB-medium supplemented with 20 mM MgCl₂ and carbenicillin50 μg/ml and redissolved in PBS after harvesting. By four rounds offreezing at −70° C. and thawing at 37° C., the outer membrane of thebacteria was destroyed by osmotic shock and the soluble periplasmicproteins including the scFvs were released into the supernatant. Afterelimination of intact cells and cell-debris by centrifugation, thesupernatant containing the human anti-human CD3-scFvs was collected andused for further examination. These crude supernatants containing scFvwill be further termed periplasmic preparations (PPP).

23.2 Binding of scFvs to Human CD3 Epsilon (aa 1-27)-Fc Fusion Protein

ELISA experiments were carried out by coating the human CD3 epsilon (aa1-27)-Fc fusion protein to the wells of 96 well plastic plates (Nunc,maxisorb) typically at 4° C. over night. The antigen coating solutionwas then removed, wells washed once with PBS/0.05% Tween 20 andsubsequently blocked with PBS/3% BSA for at least one hour. Afterremoval of the blocking solution, PPPs and control solutions were addedto the wells and incubated for typically one hour at room temperature.The wells were then washed three times with PBS/0.05% Tween 20.Detection of scFvs bound to immobilized antigen was carried out using aBiotin-labeled anti FLAG-tag antibody (M2 anti Flag-Bio, Sigma,typically at a final concentration of 1 μg/ml PBS) and detected with aperoxidase-labeled Streptavidine (Dianova, 1 μg/ml PBS). The signal wasdeveloped by adding ABTS substrate solution and measured at a wavelengthof 405 nm. Unspecific binding of the test-samples to the blocking agentand/or the human IgG1 portion of the human CD3 epsilon (aa 1-27)-Fcfusion protein was examined by carrying out the identical assay with theidentical reagents and identical timing on ELISA plates which werecoated with human IgG1 (Sigma). PBS was used as a negative control.

As shown in FIG. 44, scFvs H2C, F12Q and I2C show strong binding signalson human CD3 epsilon (aa 1-27)-Fc fusion protein. The human scFvs 3-106,3-114, 3-148, 3-190, 3-271, 4-10 and 4-48 (as described in WO2004/106380) do not show any significant binding above negative controllevel.

To exclude the possibility that the positive binding of scFvs H2C, F12Qand I2C to wells coated with human CD3 epsilon (aa 1-27)-Fc fusionprotein might be due to binding to BSA (used as a blocking agent) and/orthe human IgG1 Fc-gamma-portion of the human CD3 epsilon (aa 1-27)-Fcfusion protein, a second ELISA experiment was performed in parallel. Inthis second ELISA experiment, all parameters were identical to those inthe first ELISA experiment, except that in the second ELISA experimenthuman IgG1 (Sigma) was coated instead of human CD3 epsilon (aa 1-27)-Fcfusion protein. As shown in FIG. 45, none of the scFvs tested showed anysignificant binding to BSA and/or human IgG1 above background level.

Taken together, these results allow the conclusion that conventional CD3binding molecules recognizing a context-dependent epitope of CD3 epsilon(e.g. as disclosed in WO 2004/106380) do not bind specifically to thehuman CD3 epsilon (aa 1-27)-region, whereas the scFvs H2C, F12Q and I2Cbinding a context-independent epitope of CD3 epsilon clearly showspecific binding to the N-terminal 27 amino acids of human CD3 epsilon.

24. Generation and Characterization of Single Domain EGFR and CD3Cross-Species Specific Bispecific Single Chain Molecules

24.1 Generation of CHO Cells Transfected with Human EGFR

The cell line positive for human EGFR, A431 (epidermoid carcinoma cellline, CRL-1555, American Type Culture Collection, Rockville, Md.) wasused to obtain total RNA that was isolated according to the instructionsof the kit manual (Qiagen, RNeasy Mini Kit, Hilden, Germany). Theobtained RNA was used for cDNA synthesis by random-primed reversetranscription. For cloning of the full length sequence of the human EGFRantigen the following oligonucleotides were used:

5′ EGFR AG XbaI (SED ID NO 402) 5′-GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG-3′3′ EGFR AG SalI (SEQ ID NO 403)5′-TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT-3′

The coding sequence was amplified by PCR (denaturation at 94° C. for 5min, annealing at 58° C. for 1 min, elongation at 72° C. for 2 min forthe first cycle; denaturation at 94° C. for 1 min, annealing at 58° C.for 1 min, elongation at 72° C. for 2 min for 30 cycles; terminalextension at 72° C. for 5 min). The PCR product was subsequentlydigested with XbaI and SalI, ligated into the appropriately digestedexpression vector pEF-DHFR (Raum et al., Cancer Immunol. Immunother.2001; 50: 141-150), and transformed into E. coli. The afore-mentionedprocedures were carried out according to standard protocols (Sambrook,Molecular Cloning; A Laboratory Manual, 3rd edition, Cold Spring HarbourLaboratory Press, Cold Spring Harbour, N.Y. (2001)). A clone withsequence-verified nucleotide sequence (SEQ ID 370, Amino acid sequenceSEQ ID 369) was transfected into DHFR deficient CHO cells for eukaryoticexpression of the construct. Eukaryotic protein expression in DHFRdeficient CHO cells was performed as described by Kaufmann R. J. (1990)Methods Enzymol. 185, 537-566. Gene amplification of the construct wasinduced by increasing concentrations of methothrexate (MTX) to a finalconcentration of up to 20 nM MTX.

24.2 Generation of CHO Cells Expressing the Extracellular Domain ofMacaque EGFR

The cDNA sequence of the extracellular domain of macaque EGFR wasobtained by a set of two PCRs on macaque monkey colon cDNA (Cat#:C1534090-Cy-BC; obtained from BioCat GmbH, Heidelberg, Germany) usingthe following reaction conditions: 1 cycle at 94° C. for 3 minutesfollowed by 35 cycles with 94° C. for 1 minute, 53° C. for 1 minute and72° C. for 2 minutes followed by a terminal cycle of 72° C. for 3minutes. The following primers were used:

4. forward primer: (SEQ ID NO 404) 5′-CGCTCTGCCCGGCGAGTCGGGC-3′ reverseprimer: (SEQ ID NO 405) 5′-CCGTCTTCCTCCATCTCATAGC-3′ 5. forward primer:(SEQ ID NO 406) 5′-ACATCCGGAGGTGACAGATCACGGCTCGTGC-3′ reverse primer:(SEQ ID NO 407) 5′-CAGGATATCCGAACGATGTGGCGCCTTCGC-3′

Those PCRs generated two overlapping fragments (A: 1-869, B: 848-1923),which were isolated and sequenced according to standard protocols usingthe PCR primers, and thereby provided a 1923 bp portion of the cDNAsequence of macaque EGFR from the third nucleotide of codon +1 of themature protein to the 21^(st) codon of the transmembrane domain. Togenerate a construct for expression of macaque EGFR a cDNA fragment wasobtained by gene synthesis according to standard protocols (the cDNA andamino acid sequence of the construct is listed under SEQ ID Nos 372 and371). In this construct the coding sequence for macaque EGFR from aminoacid +2 to +641 of the mature EGFR protein was fused into the codingsequence of human EGFR replacing the coding sequence of the amino acids+2 to +641. The gene synthesis fragment was also designed as to containa Kozak site for eukaryotic expression of the construct and restrictionsites at the beginning and the end of the cDNA coding for essentiallythe extracellular domain of macaque EGFR fused to the transmembrane andintracellular domains of human EGFR. Furthermore a conservative mutationwas introduced at amino acid 627 (4^(th) amino acid of the transmembranedomain) mutating valine into leucine to generate a restriction site(SphI) for cloning purposes. The introduced restriction sites XbaI atthe 5′ end and SalI at the 3′ end, were utilised in the followingcloning procedures. The gene synthesis fragment was then cloned via XbaIand SalI into a plasmid designated pEF-DHFR (pEF-DHFR is described inMack et al. Proc. Natl. Acad. Sci. USA 92 (1995) 7021-7025). A sequenceverified clone of this plasmid was used to transfect CHO/dhfr-cells asdescribed above.

24.3 Cloning of Cross-Species Specific Single Domain Binding Molecules

Generally, bispecific single chain antibody molecules, each comprising adomain with a binding specificity cross-species specific for human andnon-chimpanzee primate CD3 epsilon as well as a domain with a bindingspecificity cross-species specific for human and non-chimpanzee primateEGFR, were designed as set out in the following Table 7:

TABLE 7 Formats of anti-CD3 and anti-EGFR cross-species specific singledomain bispecific single chain antibody molecules SEQ ID Formats ofprotein constructs (nucl/prot) (N → C) 381/380 EGFR 3D-E8 × I2C HL385/384 EGFR 3D-E8 × F12Q HL 383/382 EGFR 3D-E8 × H2C HL 392/391 EGFR3D-D12 × I2C HL 396/395 EGFR 3D-D12 × F12Q HL 394/393 EGFR 3D-D12 × H2CHL

The aforementioned constructs containing the variable chaincross-species specific for human and macaque EGFR and CD3 were obtainedby gene synthesis. The gene synthesis fragments were designed as tocontain first a Kozak site for eukaryotic expression of the construct,followed by a 19 amino acid immunoglobulin leader peptide, followed inframe by the coding sequence of the respective bispecific single chainantibody molecule, followed in frame by the coding sequence of a 6histidine tag and a stop codon. The gene synthesis fragment was alsodesigned as to introduce suitable restriction sites at the beginning andat the end of the fragment. The introduced restriction sites wereutilized in the following cloning procedures. The gene synthesisfragment was also designed as to introduce suitable N- and C-terminalrestriction sites. The gene synthesis fragment was cloned via theserestriction sites into a plasmid designated pEF-DHFR (pEF-DHFR isdescribed in Raum et al. Cancer Immunol Immunother 50 (2001) 141-150)according to standard protocols (Sambrook, Molecular Cloning; ALaboratory Manual, 3rd edition, Cold Spring Harbour Laboratory Press,Cold Spring Harbour, N.Y. (2001)). A clone with sequence-verifiednucleotide sequence was transfected into dihydrofolate reductase (DHFR)deficient Chinese hamster ovary (CHO) cells for eukaryotic expression ofthe construct.

The constructs were transfected stably or transiently intoDHFR-deficient CHO-cells (ATCC No. CRL 9096) by electroporation oralternatively into HEK 293 (human embryonal kidney cells, ATCC Number:CRL-1573) in a transient manner according to standard protocols.

24.4. Expression and Purification of the Single Domain Bispecific SingleChain Antibody Molecules

The single domain bispecific single chain antibody molecules wereexpressed in chinese hamster ovary cells (CHO). Eukaryotic proteinexpression in DHFR deficient CHO cells was performed as described byKaufmann R. J. (1990) Methods Enzymol. 185, 537-566. Gene amplificationof the constructs was induced by increasing final concentrations of MTXup to 20 nM. After two passages of stationary culture the cells weregrown in roller bottles with nucleoside-free HyQ PF CHO liquid soymedium (with 4.0 mM L-Glutamine with 0.1% Pluronic F-68; HyClone) for 7days before harvest. The cells were removed by centrifugation and thesupernatant containing the expressed protein was stored at −20° C.Alternatively, constructs were transiently expressed in HEK 293 cells.Transfection was performed with 293fectin reagent (Invitrogen,#12347-019) according to the manufacturer's protocol.

Äkta® Explorer System (GE Health Systems) and Unicorn® Software wereused for chromatography. Immobilized metal affinity chromatography(“IMAC”) was performed using a Fractogel EMD chelate® (Merck) which wasloaded with ZnCl2 according to the protocol provided by themanufacturer. The column was equilibrated with buffer A (20 mM sodiumphosphate buffer pH 7.2, 0.1 M NaCl) and the cell culture supernatant(500 ml) was applied to the column (10 ml) at a flow rate of 3 ml/min.The column was washed with buffer A to remove unbound sample. Boundprotein was eluted using a two step gradient of buffer B (20 mM sodiumphosphate buffer pH 7.2, 0.1 M NaCl, 0.5 M Imidazol) according to thefollowing:

Step 1: 20% buffer B in 6 column volumesStep 2: 100% buffer B in 6 column volumes

Eluted protein fractions from step 2 were pooled for furtherpurification. All chemicals were of research grade and purchased fromSigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration chromatography was performed on a HiLoad 16/60 Superdex200 prep grade column (GE/Amersham) equilibrated with Equi-buffer (25 mMCitrat, 200 mM Lysin, 5% Glycerol, pH 7.2). Eluted protein samples (flowrate 1 ml/min) were subjected to standard SDS-PAGE and Western Blot fordetection. Prior to purification, the column was calibrated formolecular weight determination (molecular weight marker kit, Sigma MWGF-200). Protein concentrations were determined using OD280 nm.

Purified bispecific single chain antibody protein was analyzed in SDSPAGE under reducing conditions performed with pre-cast 4-12% Bis Trisgels (Invitrogen). Sample preparation and application were performedaccording to the protocol provided by the manufacturer. The molecularweight was determined with MultiMark protein standard (Invitrogen). Thegel was stained with colloidal Coomassie (Invitrogen protocol). Thepurity of the isolated protein was >95% as determined by SDS-PAGE.

Western Blot was performed using an Optitran® BA-S83 membrane and theInvitrogen Blot Module according to the protocol provided by themanufacturer. The antibodies used were directed against the His Tag(Penta His, Qiagen) and Goat-anti-mouse Ig labeled with alkalinephosphatase (AP) (Sigma), and BCIP/NBT (Sigma) as substrate.

24.5. Flow Cytometric Binding Analysis of the EGFR and CD3 Cross-SpeciesSpecific Single Domain Bispecific Antibodies

In order to test the functionality of the single domain cross-speciesspecific bispecific antibody constructs with regard to bindingcapability to human and macaque EGFR and CD3, respectively, a FACSanalysis was performed. For this purpose CHO cells transfected withhuman EGFR as described in Example 24.1 and human CD3 positive T cellleukemia cell line Jurkat (DSMZ, Braunschweig, ACC 282) were used totest the binding to human antigens. The binding reactivity to macaqueantigens was tested by using the generated macaque EGFR transfectantdescribed in Example 24.2 and a macaque T cell line 4119LnPx (kindlyprovided by Prof Fickenscher, Hygiene Institute, Virology,Erlangen-Nuernberg; published in Knappe A, et al., and Fickenscher H.,Blood 2000, 95, 3256-61). 200.000 cells of the respective cellpopulation were incubated for 30 min on ice with 50 μl of the purifiedprotein of the cross-species specific bispecific antibody constructs (2μg/ml). Alternatively, the cell culture supernatant of transientlyproduced proteins was used. The cells were washed twice in PBS andbinding of the construct was detected with a murine Penta His antibody(Qiagen; diluted 1:20 in 50 μl PBS with 2% FCS). After washing, boundanti His antibodies were detected with an Fc gamma-specific antibody(Dianova) conjugated to phycoerythrin, diluted 1:100 in PBS with 2% FCS.Fresh culture medium was used as a negative control.

Flow cytometry was performed on a FACS-Calibur apparatus, the CellQuestsoftware was used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

The binding ability of several single domain bispecific single chainmolecules which are specific for EGFR and cross-species specific forhuman and non-chimpanzee primate CD3 were clearly detectable as shown inFIG. 46/1 and FIG. 46/2 In the FACS analysis, all constructs showedbinding to CD3 and EGFR compared to culture medium and first and seconddetection antibody as the negative controls. Cross-species specificityof the bispecific antibody to human and macaque CD3 and EGFR antigenswas demonstrated.

24.6. Bioactivity of EGFR and CD3 Cross-Species Specific Single DomainBispecific Single Chain Antibodies

Bioactivity of the generated single domain bispecific single chainantibodies was analyzed by chromium 51 (⁵¹Cr) release in vitrocytotoxicity assays using the EGFR positive cell lines described inExamples 24.1 and 24.2. As effector cells stimulated human CD8 positiveT cells or the macaque T cell line 4119LnPx were used, respectively.

Stimulated CD8+ T cells were obtained as follows:

A Petri dish (145 mm diameter, Greiner) was pre-coated with acommercially available anti-CD3 specific antibody in a finalconcentration of 1 μg/ml for 1 hour at 37° C. Unbound protein wasremoved by one washing step with PBS. The fresh PBMC's were isolatedfrom peripheral blood (30-50 ml human blood) by Ficoll gradientcentrifugation according to standard protocols. 3-5×10⁷ PBMCs were addedto the precoated petri dish in 120 ml of RPMI 1640/10% FCS/IL-2 20 U/ml(Proleukin, Chiron) and stimulated for 2 days. At the third day thecells were collected, washed once with RPMI 1640. IL-2 was added to afinal concentration of U/ml and cultivated again for one day. CD8+cytotoxic T lymphocytes (CTLs) were isolated by depletion of CD4+ Tcells and CD56+ NK cells.

Target cells were washed twice with PBS and labeled with 11.1 MBq ⁵¹Crin a final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37° C.Subsequently the labeled target cells were washed 3 times with 5 ml RPMIand then used in the cytotoxicity assay. The assay was performed in a 96well plate in a total volume of 250 μl supplemented RPMI (as above) withan E:T ratio of 10:1. 1 μg/ml of the cross-species specific bispecificsingle chain antibody molecules and 20 threefold dilutions thereof wereapplied. Alternatively cell culture supernatant of transiently producedproteins was serially diluted in 1:2 steps. The assay time is 18 hoursand cytotoxicity was measured as relative values of released chromium inthe supernatant related to the difference of maximum lysis (addition ofTriton-X) and spontaneous lysis (without effector cells). Allmeasurements were done in quadruplicates. Measurement of chromiumactivity in the supernatants was performed with a Wizard 3″ gammacounter(Perkin Elmer Life Sciences GmbH, KoIn, Germany). Analysis of theexperimental data was performed with Prism 4 for Windows (version 4.02,GraphPad Software Inc., San Diego, Calif., USA). Sigmoidal dose responsecurves typically had R2 values >0.90 as determined by the software. EC50values calculated by the analysis program were used for comparison ofbioactivity.

As shown in FIGS. 47/1 and FIG. 47/2 all of the generated single domaincross-species specific bispecific single chain antibody constructsrevealed cytotoxic activity against human EGFR positive target cellselicited by human CD8+ cells and macaque EGFR positive target cellselicited by the macaque T cell line 4119LnPx. A bispecific single chainantibody with different target specificity was used as negative control.

25 Generation of Cross-Species Specific Bispecific Single ChainMolecules Comprising One or Two Single-Domain Antibody Binders 25 A)Generation of Cross-Species Specific Single Domain Antibodies Binding tothe N-Terminal Amino Acids 1-27 of CD3Epsilon of Man and DifferentNon-Chimpanzee Primates 25 A1) Preparation of Peptide Conjugates of theN-Terminal Amino Acids 1-27 of CD3Epsilon of Man to KLH and BSA forImmunization

18 mg of a lyophilized peptide of the sequenceacetyl-QDGNEEMGGITQTPYKVSISGTTVILTC (the additional amino acid “C” atthe C-terminal position is added for coupling the 1-27 amino acidconstruct to e.g. KLH; SEQ ID NO 425) obtained by peptide synthesisaccording to standard protocols was dissolved in Dimethylformamide (DMF)and then diluted in phosphate buffered saline with 1 mMethylenediaminetetraacetic acid (PBS/EDTA) resulting in a finalconcentration of 10% DMF to keep the peptide dissolved. This peptidesolution was applied onto a Reduce-Imm™ Reducing Column (Pierce,Rockford Ill.). Activation and washing of the column as well as allsubsequent steps were performed according to the protocol of themanufacturer. The peptide solution was applied to the column and elutedresulting in the reductive cleavage of the disulphide bond between theC-terminal cysteines of two peptides generating a free cysteine on everypeptide. One half of the reduced peptide solution was mixed with 6 mg ofmaleimide activated bovine serum albumin BSA (Pierce, Rockford Ill.).The remaining peptide solution was mixed with 6 mg of maleimideactivated keyhole limpet hemocyanin KLH (Pierce, Rockford Ill.).Conjugation was performed for 2 hours at room temperature underagitation and protection from light. Afterwards both conjugates weredialyzed three times against PBS according to standard protocols toachieve a physiological formulation.

25 A2) Immunization of Camelids Using the N-Terminus of CD3 EpsilonSeparated from its Native CD3-Context by Fusion to a HeterologousSoluble Protein as Well as Conjugation to KLH

1 to 8 years old Alpaca/Llama crossings were immunized with theCD3epsilon-Fc fusion protein carrying the most N-terminal amino acids1-27 of the mature CD3epsilon chain (1-27 CD3-Fc) of man, theCD3epsilon-Fc fusion protein carrying the most N-terminal amino acids1-27 of the mature CD3epsilon chain (1-27 CD3-Fc) of saimiri sciureus, apeptide-BSA conjugate of the most N-terminal amino acids 1-27 of themature CD3epsilon chain (1-27 CD3-Fc) of man and a peptide-KLH conjugateof the most N-terminal amino acids 1-27 of the mature CD3epsilon chain(1-27 CD3-Fc) of man all as described above. To this end for each animal500 μg of a 1:1 mixture of the two 1-27 CD3-Fc fusion proteins in atotal volume of 2 ml PBS were mixed with 1 ml complete Freund's adjuvantand injected subcutaneously. The animals received booster immunizationsafter 28 and 42 days and optionally also after 56, 70 and 84 days. Thefirst booster immunization was performed with 500 μg of a 1:1 mixture ofthe two 1-27 CD3-Fc fusion proteins in a total volume of 2 ml PBS mixedwith 1 ml incomplete Freund's adjuvant and injected subcutaneously. Thesecond and all following booster immunizations were performedalternately with 500 μg of peptide-KLH conjugate and peptide-BSAconjugate described above diluted with PBS to a final volume of 2 mlmixed with 1 ml incomplete Freund's adjuvant and injectedsubcutaneously.

52 days after the first immunization, blood samples were taken andantibody serum titers against the CD3-positive human T cell line HPBaIIand the macaque CD3-positive T cell line 4119LnPx (kindly provided byProf Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg;published in Knappe A, et al., and Fickenscher H., Blood 2000, 95,3256-61) were tested in flow cytometry according to standard protocols.To this end 200.000 cells of the respective cell lines were incubatedfor 30 min on ice with 50 μl of serum of the immunized animals diluted1:1000 in PBS with 2% FCS. The cells were washed twice in PBS with 2%FCS and binding of serum antibodies was detected with a FITC conjugatedGoat anti-Llama IgG-H&L Antibody (Bethyl Laboratories Inc., Catalog No.A160-100F) diluted 1:100 in 50 μl PBS with 2% FCS. Serum of the animalsobtained prior to immunization was used as a negative control.

Flow cytometry was performed on a FACS-Calibur apparatus, the CellQuestsoftware was used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity were performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

Reactivity to the CD3-positive human T cell line HPBaII and theCD3-positive macaque T cell line 4119LnPx of a serum sample of oneexemplary animal obtained 52 days after the first immunization wasclearly detectable as shown in FIG. 48.

25 A3) Generation of an Immune Camelid Single Domain Antibody Library:Construction of an Antibody Library and Phage Display

Hundred milliliter of peripheral blood were obtained from each of threeanimals with antigen-positive serum titers. Peripheral blood mononuclearcells (PBMCs) were isolated by a ficoll-gradient according to standardmethods. Total RNA was isolated from the PBMCs using the RNeasy® MidiKit (QIAGEN) following the manufacturer's instructions. cDNA wassynthesized according to standard methods (Sambrook, Cold Spring HarborLaboratory Press 1989, Second Edition).

For the isolation of V-region DNA, RT-PCR was carried out using aspecific primer set consisting of the 5′ primers:

5′-VHHa-XhoI: (SEQ ID NO 408) 5′-CTG ACG CTC GAG GAG GTG CAG CTG GTG GAGTCT GG-3′, 5′-VHHb-XhoI: SEQ ID NO 409) 5′-CTG ACG CTC GAG CAG GTR CAGCTG GTG GAG TCT GG-3′ 5′-VHHc-XhoI: (SEQ ID NO 410) 5′-CTG ACG CTC GAGCAG GTA AAG CTG GAG GAG TCT GG-3′ 5′-VHHd-XhoI: (SEQ ID NO 411) 5′-CTGACG CTC GAG GAT GTG CAG CTG GTG GAG TCT GG-3′ 5′-VHHe-XhoI: (SEQ ID NO412) 5′-CTG ACG CTC GAG GCC GTG CAG CTG GTG GAT TCT GG-3′ 5′-VHHf-XhoI:(SEQ ID NO 413) 5′-CTG ACG CTC GAG GCG GTG CAG CTG GTG GAG TCT GG-3′5′-VHH-LP-A-XhoI: (SEQ ID NO 414) 5′-CTG ACG CTC GAG GAG GTG CAG CTG CAGGCG TCT G-3′ 5′-VHH-LP-B-XhoI: (SEQ ID NO 415) 5′-CTG ACG CTC GAG GATGTS CAG CTG CAG GCG TCT G-3′ 5′-VHH-LX-I-XhoI: (SEQ ID NO 416) 5′-CTGACG CTC GAG CAG GTG CAG CTG GTG CAG TCTGG-3′ 5′-VHH-LX-II-XhoI: (SEQ IDNO 417) 5′-CTG ACG CTC GAG CAG GTC ACC TTG AAG GAG TCTGG-3′5′-VHH-LX-III-XhoI: (SEQ ID NO 418) 5′-CTG ACG CTC GAG CAG GTG CAG CTGCAG GAG TCGGG-3′ 5′-VHH-LG-1-XhoI: (SEQ ID NO 419) 5′-CTG ACG CTC GAGCTG CAG CAG TCT GGG GGA GG-3′and of the 3′-primers:

3′-VHHG2-BsiWI-SpeI: (SEQ ID NO 420) 5′-CTG ACG ACT AGT CGT ACG TTG GGGTAT CTT GGG TTC TG-3′ 3′-VHHG3-BsiWI-SpeI: (SEQ ID NO 421) 5′-CTG ACGACT AGT CGT ACG TAC TTC ATT CGT TCC TGA VGA G-3′3′-VHH-LP-G2a-BsiWI-SpeI: (SEQ ID NO 422) 5′-CTG ACG ACT AGT CGT ACG TTGTGG TTT TGG TGT CTT GGG TTC-3′ 3′-VHH-LP-dirA-BsiWI-SpeI: (SEQ ID NO423) 5′-CTG ACG ACT AGT CGT ACG TGA GGA GAC GGT GAC CTG GGT CC-3′3′-VHH-LG-dir1-BsiWI-SpeI: (SEQ ID NO 424) 5′-CTG ACG ACT AGT CGT ACGGGT GAC CTG GGT CCC CTG GC-3′

The primers chosen for PCR amplification gave rise to 5′-XhoI and3′-SpeI recognition sites for the V chain fragments. Per PCR reaction,one specific 5′-primer was combined with one specific 3′-primer. Thenumber of different PCR reactions was determined by the number ofpossible combinations of 5′- and 3′-primers. The following PCR-programwas used for amplification: Denaturation at 94° C. for 15 seconds,primer annealing at 52° C. for 50 seconds and primer extension at 72° C.for 90 seconds were performed over 40 cycles, followed by finalextension at 72° C. for 10 minutes. DNA V-fragments were then isolatedaccording to standard protocols. 300 ng of the V chain fragments(XhoI-SpeI digested) were ligated with 1400 ng of the phagemidpComb3H5Bhis (XhoI-SpeI digested; large fragment). The resultingantibody library was then transformed into 300 ul of electrocompetentEscherichia coli XL1 Blue cells by electroporation (2.5 kV, 0.2 cm gapcuvette, 25 uFD, 200 Ohm, Biorad gene-pulser) resulting in a librarysize of more than 10E7 independent clones. After one hour of phenotypeexpression and slow adaptation to carbenicillin, the E. coli cellscontaining the antibody library were transferred into SB-Carbenicillin(50 ug/mL) selection medium. The E. coli cells containing the antibodylibrary were then infected with an infectious dose of 10E12 particles ofhelper phage VCSM13 resulting in the production and secretion offilamentous M13 phage. The resulting library comprises phage particles,which contain single stranded pComb3H5BHis-DNA encoding a camelidV-fragment and display the corresponding V-protein as a translationalfusion to phage coat protein III. This pool of phages displaying theantibody library was later used for the selection of antigen bindingentities.

25 A4) Phage Display Based Selection of CD3-Specific Single-DomainBinders

The phage library carrying the cloned V-repertoire was harvested fromthe respective culture supernatant by PEG8000/NaCl precipitation andcentrifugation. Approximately 10E11 to 10E12 scFv phage particles wereresuspended in 0.4 ml of PBS/0.1% BSA and incubated with 10E5 to 10E7HPBaII cells (a CD3-positive human T-cell line) for 1 hour on ice underslow agitation. These Jurkat cells were grown beforehand in RPMI mediumenriched with fetal calf serum (10%), glutamine andpenicillin/streptomycin, harvested by centrifugation, washed in PBS andresuspended in PBS/1% FCS (containing Na Azide). Phage particles whichdo not specifically bind to the Jurkat cells via the displayed V domainwere eliminated by up to five washing steps with PBS/1% FCS (containingNa Azide). After washing, specifically bound phages were eluted from thecells by resuspending the cells in HCl-glycine pH 2.2 (10 min incubationwith subsequent vortexing) and after neutralization with 2 M Tris pH 12,the eluate was used for infection of a fresh uninfected E. coli XL1 Blueculture (OD600 >0.5). The E. coli culture containing E. coli cellssuccessfully transduced with a phagemid copy, encoding a V-fragment,were again selected for carbenicillin resistance and subsequentlyinfected with VCMS 13 helper phage to start the second round of antibodydisplay and in vitro selection. A total of 4 to 5 rounds of selectionswere carried out. Every second round of selection was performed usingmacaque T cell line 4119LnPx (kindly provided by Prof Fickenscher,Hygiene Institute, Virology, Erlangen-Nuernberg; published in Knappe A,et al., and Fickenscher H., Blood 2000, 95, 3256-61) instead of Jurkatcells.

25 A5) Screening for CD3-Specific Single-Domain Binders

Plasmid DNA corresponding to 4 and 5 rounds of panning was isolated fromE. coli cultures after selection. For the production of soluble Vprotein, V DNA fragments were excised from the plasmids (XhoI-SpeI).These fragments were cloned via the same restriction sites into theplasmid pComb3H5BFlag/His differing from the original pComb3H5BHis inthat the expression construct (i.e. V domain) includes a Flag-tag (TGDYKDDDDK) at ot's C-terminus before the His6-tag and that phage proteinIII/N2 domain and protein III/CT had been deleted. After ligation, eachpool (different rounds of panning) of plasmid DNA was transformed into100 μl heat shock competent E. coli TG1 or XLI blue and plated ontocarbenicillin LB-agar. Single colonies were picked into 100 ul of LBcarb (50 ug/ml).

E. coli transformed with pComb3H₅BHis containing a V-segment producesoluble V domains in sufficient amounts after excision of the gene IIIfragment and induction with 1 mM IPTG. Due to a suitable signalsequence, the V-chain was exported into the periplasma where it foldsinto a functional conformation.

Single E. coli TG1 bacterial colonies from the transformation plateswere picked for periplasmic small scale preparations and grown inSB-medium (e.g. 10 ml) supplemented with 20 mM MgCl2 and carbenicillin50 μg/ml (and re-dissolved in PBS (e.g. 1 ml) after harvesting. By fourrounds of freezing at −70° C. and thawing at 37° C., the outer membraneof the bacteria was destroyed by temperature shock and the solubleperiplasmic proteins including the V domains were released into thesupernatant.

After elimination of intact cells and cell-debris by centrifugation, thesupernatant containing the anti-CD3 single-domain antibodies wascollected and used for further examination.

25 A6) Identification of CD3-Specific Single-Domain Binders

Binding of the isolated single-domain antibodies to human andnon-chimpanzee primate CD3 was tested by flowcytometry on the human CD3positive T cell leukemia cell line HPB-ALL (DSMZ, Braunschweig, ACC483)and the CD3 positive macaque T cell line 4119LnPx (kindly provided byProf Fickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg;published in Knappe A, et al., and Fickenscher H., Blood 2000, 95,3256-61).

For flow cytometry 2.5×10⁵ cells are incubated with 50 ul supernatant orwith 5 μg/ml of the purified constructs in 50 μl PBS with 2% FCS. Thebinding of the constructs was detected with an anti-His antibody(Penta-His Antibody, BSA free, Qiagen GmbH, Hilden, FRG) at 2 μg/ml in50 μl PBS with 2% FCS. As a second step reagent aR-Phycoerythrin-conjugated affinity purified F(ab′)2 fragment, goatanti-mouse IgG (Fc-gamma fragment specific), diluted 1:100 in 50 μl PBSwith 2% FCS (Dianova, Hamburg, FRG) was used. The samples were measuredon a FACSscan (BD biosciences, Heidelberg, FRG). FIG. 49 showscross-species specific binding of anti-CD3 single domain binder CD33D-H11 to human and macaque T cells and no binding to CD3 negative CHOcells.

25 A7) Binding Assay of Cross-Species Specific Single Chain Antibodiesto the N-Terminus of CD3Epsilon (Amino Acids 1-27) Separated from itsNative CD3-Context in a Soluble Fc-Fusion Protein and Conjugated as27mer-Petide to BSA.

Binding of crude preparations of periplasmatically expressed anti-CD3single domain antibody CD3 3D-H11 to immobilized 1-27 CD3-Fc fusionprotein and 1-27 CD3 BSA conjugate was tested in an ELISA assay. Antigenimmobilization was carried out by overnight incubation of 5 μg/mlantigen in PBS at 4° C. Wells were washed with PBS containing 0.05%Tween 20 (PBS/Tween) and blocked with PBS containing 3% BSA (bovineAlbumin, fraction V, Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany)for 60 minutes at RT. Subsequently, wells were incubated with crudepreparations of periplasmatically expressed single domain antibody asdescribed above for 60 minutes at room temperature. After washing withPBS/Tween wells were incubated with peroxidase conjugated anti-Flag M2antibody (Sigma-Aldrich Chemie GmbH, Taufkirchen, Germany) diluted1:10000 in PBS with 1% BSA for 60 minutes at RT. Wells were washed withPBS/Tween and incubated with 100 μl of the SIGMAFAST OPD (OPD[o-Phenylenediamine dihydrochloride] substrate solution (Sigma-AldrichChemie GmbH, Taufkirchen, Germany) according to the manufacturersprotocol. Color reaction was stopped with 100 μl 1 M H₂SO₄ and measuredon a PowerWaveX microplate spectrophotometer (BioTek Instruments, Inc.,Winooski, Vt., USA) at 490 nm and subtraction of background absorptionat 620 nm. Binding of anti-CD3 single domain antibody H11 to 1-27 CD3-Fcfusion protein as well as to 1-27 CD3 BSA conjugate is shown in FIG. 50

25 A8) Generation of Human/Humanized Equivalents of Non-Human CD3EpsilonSpecific Single-Domain Binders

The camelid V region was aligned against human antibody germline aminoacid sequences. The human antibody germline VH sequence was chosen whichhas the closest homology to the non-human V domain and a directalignment of the two amino acid sequences was performed. There were anumber of framework residues of the non-human V domain that differ fromthe human VH framework regions (“different framework positions”). Someof these residues may contribute to the binding and activity of theantibody to its target.

To construct a library that contains the non-human CDRs and at everyframework position that differs from the chosen human VH sequence bothpossibilities (the human and the non-human amino acid residue),degenerated oligonucleotides were synthesized. These oligonucleotidesincorporate at the differing positions the human residue with aprobability of 75% and the non-human residue with a probability of 25%.For one human V domain e.g. six of these oligonucleotides had to besynthesized that overlap in a terminal stretch of approximately 20nucleotides. To this end every second primer was an antisense primer.Restriction sites needed for later cloning of the V region had to beavoided within the nucleotide sequence of these oligonucleotides, e.g.by means of silent (i.e. amino acid neutral) nucleotide exchange ifrequired.

These primers may have a length of 60 to 90 nucleotides, depending onthe number of primers needed to span over the whole V sequence.

These e.g. six primers were mixed in equal amounts (e.g. 1 μl of eachprimer (primer stocks 20 to 100 μM) to a 20 μl PCR reaction) and addedto a PCR mix consisting of PCR buffer, nucleotides and Taq polymerase.This mix was incubated at 94° C. for 3 minutes, 65° C. for 1 minute, 62°C. for 1 minute, 59° C. for 1 minute, 56° C. for 1 minute, 52° C. for 1minute, 50° C. for 1 minute and at 72° C. for 10 minutes in a PCRcycler. Subsequently the product was run in an agarose gelelectrophoresis and the product of a size from 200 to 400 isolated fromthe gel according to standard methods.

This PCR product was then used as a template for a standard PCR reactionusing primers that incorporate N-terminal and C-terminal suitablecloning restriction sites. The DNA fragment of the correct size (for a Vdomain approximately 350 nucleotides) was isolated by agarose gelelectrophoresis according to standard methods. In this way sufficient Vdomain DNA fragment was amplified. This V domain DNA fragment containeda pool of human-like V domains differing from each other by the numberof human and non-human residues at the framework positions whichoriginally differed between the human and the non-human V region.

The pool of human-like V domains was then cloned into the phage displayvector pComb3H5Bhis to form a library of functional V domains fromwhich—after display on filamentous phage—anti-CD3 binders were selected,screened, identified and confirmed as described above for the parentalnon-human (camelid) anti-CD3 V domain. Single clones were then analyzedfor favorable properties and amino acid sequence. Those V domains whichwere closest in amino acid sequence homology to human germlineV-segments are preferred.

Anti-CD3 single-domain binders are converted into recombinant bispecificsingle chain antibodies and further characterized as follows.

25 B) Generation and Characterization of Cross-Species SpecificBispecific Single Chain Molecules Comprising One or Two Single-DomainAntibody Binders 25 B1) Construction and Expression of Bispecific SingleChain Molecules Comprising One or Two Single-Domain Antibody Binders

Anti-CD3 single-domain binders are converted into recombinant bispecificsingle chain antibodies by joining them via a Gly4Ser1-linker with atarget antigen specific single domain (V) or scFv-binder (VH-VL orVL-VH) to result in constructs with the following arrangements of threeor two domains: VH_(Target)-(Gly₄Ser_(t))₃-VL_(Target)-Gly₄Ser₁-V_(CD3),or V_(Target)-Gly₄Ser₁-V_(CD3). Alternatively, further constructs withdifferent domain arrangements likeV_(CD3)-Gly₄Ser₁-VH_(Target)-(Gly₄Ser₁)₃-VL_(Target) orV_(cD3)-Gly₄Ser₁-V_(Target) or constructs using a scFv-binder in VL-VHinstead of VH-VL orientation can be generated according to standardprotocols. For expression in CHO cells the coding sequences of (i) anN-terminal immunoglobulin heavy chain leader comprising a start codonembedded within a Kozak consensus sequence and (ii) a C-terminalHis6-tag followed by a stop codon are both attached in frame to thenucleotide sequence encoding the bispecific single chain antibodiesprior to insertion of the resulting DNA-fragment as obtained by genesynthesis into the multiple cloning site of the expression vectorpEF-DHFR (Raum et al. Cancer Immunol Immunother 50 (2001) 141-150). Aclone with sequence-verified nucleotide sequence is transfected intoDHFR deficient CHO cells for eukaryotic expression of the construct.Eukaryotic protein expression in DHFR deficient CHO cells is performedas described by Kaufmann R. J. (1990) Methods Enzymol. 185, 537-566.Gene amplification of the construct is induced by increasingconcentrations of methotrexate (MTX) to a final concentration of up to20 nM MTX.

25 B2) Production and Purification of Single Domain Bispecific SingleChain Antibody Molecules

After two passages of stationary culture CHO cells expressing singledomain bispecific single chain antibody molecules are grown in rollerbottles with nucleoside-free HyQ PF CHO liquid soy medium (with 4.0 mML-Glutamine with 0.1% Pluronic F-68; HyClone) for 7 days before harvest.The cells are removed by centrifugation and the supernatant containingthe expressed protein is stored at −20° C. Transfection is performedwith 293fectin reagent (Invitrogen, #12347-019) according to themanufacturer's protocol.

Äkta® Explorer System (GE Health Systems) and Unicorn® Software are usedfor chromatography. Immobilized metal affinity chromatography (“IMAC”)is performed using a Fractogel EMD chelate® (Merck) which is loaded withZnCl2 according to the protocol provided by the manufacturer. The columnis equilibrated with buffer A (20 mM sodium phosphate buffer pH 7.2, 0.1M NaCl) and the cell culture supernatant (500 ml) is applied to thecolumn (10 ml) at a flow rate of 3 ml/min. The column is washed withbuffer A to remove unbound sample. Bound protein is eluted using a twostep gradient of buffer B (20 mM sodium phosphate buffer pH 7.2, 0.1 MNaCl, 0.5 M Imidazol) according as follows:

Step 1: 20% buffer B in 6 column volumesStep 2: 100% buffer B in 6 column volumes

Eluted protein fractions from step 2 are pooled for furtherpurification. All chemicals can be used in research grade and purchasedfrom Sigma (Deisenhofen) or Merck (Darmstadt).

Gel filtration chromatography is performed on a HiLoad 16/60 Superdex200 prep grade column (GE/Amersham) equilibrated with Equi-buffer (25 mMCitrat, 200 mM Lysin, 5% Glycerol, pH 7.2). Eluted protein samples (flowrate 1 ml/min) are subjected to standard SDS-PAGE and Western Blot fordetection. Prior to purification, the column is calibrated for molecularweight determination (molecular weight marker kit, Sigma MW GF-200).Protein concentrations are determined using OD280 nm.

Purified bispecific single chain antibody protein is analyzed in SDSPAGE under reducing conditions performed with pre-cast 4-12% Bis Trisgels (Invitrogen). Sample preparation and application are performedaccording to the protocol provided by the manufacturer. The molecularweight is determined with MultiMark protein standard (Invitrogen). Thegel is stained with colloidal Coomassie (Invitrogen protocol). Thepurity of the isolated protein usually is >95% as determined bySDS-PAGE.

Western Blot is performed using an Optitran® BA-S83 membrane and theInvitrogen Blot Module according to the protocol provided by themanufacturer. For detection an anti-His Tag-antibody (Penta His, Qiagen)and Goat-anti-mouse Ig labeled with alkaline phosphatase (AP) (Sigma)are used; BCIP/NBT (Sigma) is used as substrate.

25 B3) Flow Cytometric Binding Analysis of Single Domain BispecificSingle Chain Antibody Molecules

In order to test the functionality of the single domain cross-speciesspecific bispecific antibody constructs with regard to bindingcapability to human and macaque target antigen and CD3, respectively, aFACS analysis is performed. For this purpose CHO cells transfected withthe human (and optionally with the macaque) target antigen, the humanCD3 positive T cell leukemia cell line Jurkat (DSMZ, Braunschweig, ACC282) and the macaque T cell line 4119LnPx (kindly provided by ProfFickenscher, Hygiene Institute, Virology, Erlangen-Nuernberg; publishedin Knappe A, et al., and Fickenscher H., Blood 2000, 95, 3256-61) areused. 200.000 cells of the respective cell population are incubated for30 min on ice with 50 μl of the purified protein of the cross-speciesspecific bispecific antibody constructs (2 μg/ml). Alternatively, thecell culture supernatant of CHO cells expressing the bispecificconstructs can be used. The cells are washed twice in PBS and binding ofthe construct is detected with a murine Penta His antibody (Qiagen;diluted 1:20 in 50 μl PBS with 2% FCS). After washing, bound anti Hisantibodies are detected with an Fc gamma-specific antibody (Dianova)conjugated to phycoerythrin, diluted 1:100 in PBS with 2% FCS. Freshculture medium is used as a negative control.

Flow cytometry is performed on a FACS-Calibur apparatus, the CellQuestsoftware is used to acquire and analyze the data (Becton Dickinsonbiosciences, Heidelberg). FACS staining and measuring of thefluorescence intensity are performed as described in Current Protocolsin Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober,Wiley-Interscience, 2002).

25. B4) Cytotoxic Activity of Single Domain Bispecific Single ChainAntibody Molecules

Bioactivity of the generated single domain bispecific single chainantibodies is analyzed by chromium 51 (⁵¹Cr) release in vitrocytotoxicity assays using target antigen transfected CHO cells and—aseffector cells—stimulated human CD8 positive T cells and the macaque Tcell line 4119LnPx.

Stimulated CD8+ T cells are obtained as follows:

A Petri dish (145 mm diameter, Greiner) is pre-coated with acommercially available anti-CD3 specific antibody in a finalconcentration of 1 μg/ml for 1 hour at 37° C. Unbound protein is removedby one washing step with PBS. The fresh PBMC's are isolated fromperipheral blood (30-50 ml human blood) by Ficoll gradientcentrifugation according to standard protocols. 3-5×10⁷ PBMCs are addedto the precoated petri dish in 120 ml of RPMI 1640/10% FCS/IL-2 20 U/ml(Proleukin, Chiron) and stimulated for 2 days. At the third day thecells are collected, washed once with RPMI 1640. IL-2 is added to afinal concentration of 20 U/ml and cultivated again for one day. CD8+cytotoxic T lymphocytes (CTLs) are isolated by depletion of CD4+ T cellsand CD56+ NK cells.

Target cells are washed twice with PBS and labeled with 11.1 MBq ⁵¹Cr ina final volume of 100 μl RPMI with 50% FCS for 45 minutes at 37° C.Subsequently the labeled target cells are washed 3 times with 5 ml RPMIand then used in the cytotoxicity assay. The assay is performed in a 96well plate in a total volume of 250 μl supplemented RPMI (as above) withan E:T ratio of 10:1. 1 μg/ml of the cross-species specific bispecificsingle chain antibody molecules and 20 threefold dilutions thereof areapplied. Alternatively cell culture supernatant of CHO cell expressingthe bispecific construct can be serially diluted in 1:2 steps. The assayduration is 18 hours and the cytotoxicity is measured as relative valuesof released chromium in the supernatant related to the difference ofmaximum lysis (addition of Triton-X) and spontaneous lysis (withouteffector cells). All measurements are usually done in quadruplicates.Measurement of released chromium activity in the supernatants isperformed with a Wizard 3″ gammacounter (Perkin Elmer Life SciencesGmbH, Köln, Germany). Analysis of the experimental data is performedwith Prism 4 for Windows (version 4.02, GraphPad Software Inc., SanDiego, Calif., USA). Sigmoidal dose response curves typically have R²values >0.90 as determined by the software. EC₅₀ values calculated bythe analysis program are used for comparison of bioactivity. Only thoseconstructs showing potent induction of T cell cytotoxicity againsttarget cells are selected for further use.

SEQ ID NO. DESIGNATION SOURCE TYPE SEQUENCE 1. Human human aaQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKE CD3εFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMD extracellular domain 2. HumanCD3ε human aa QDGNEEMGGITQTPYKVSISGTTVILT 1-27 3. Callithrix Callithrixaa QDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYjacchus CD3ε jacchus YACLSKETPAEEASHYLYLKARVCENCVEVD extracellulardomain 4. Callithrix Callithrix aa QDGNEEMGDTTQNPYKVSISGTTVTLT jacchusCD3ε jacchus 1-27 5. Saguinus Saguinus aaQDGNEEMGDTTQNPYKVSISGTTVTLTCPRYDGHEIKWLVNSQNKEGHEDHLLLEDFSEMEQSGYoedipus CD3ε oedipus YACLSKETPAEEASHYLYLKARVCENCVEVD extracellulardomain 6. Saguinus Saguinus aa QDGNEEMGDTTQNPYKVSISGTTVTLT oedipusoedipus CD3ε 1-27 7. Saimiri Saimiri aaQDGNEEIGDTTQNPYKVSISGTTVTLTCPRYDGQEIKWLVNDQNKEGHEDHLLLEDFSEMEQSGYsciureus CD3ε sciureus YACLSKETPTEEASHYLYLKARVCENCVEVD extracellulardomain 8. Saimiri Saimiri aa QDGNEEIGDTTQNPYKVSISGTTVTLT sciureussciureus CD3ε 1-27 9. CDR-L1 of F6A artificial aa GSSTGAVTSGYYPN 10.CDR-L2 of F6A artificial aa GTKFLAP 11. CDR-L3 of F6A artificial aaALWYSNRWV 12. CDR-H1 of F6A artificial aa IYAMN 13. CDR-H2 of F6Aartificial aa RIRSKYNNYATYYADSVKS 14. CDR-H3 of F6A artificial aaHGNFGNSYVSFFAY 15. VH of F6A artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSS 16. VH ofF6A artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATATCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTATCCTTCTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 17. VL of F6Aartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 18. VL of F6A artificial ntCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 19.VH-P of F6A artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSS 20. VH-P ofF6A artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATATCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTATCCTTCTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 21. VL-P of F6Aartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 22. VL-P of F6A artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 23.VH-VL of F6A artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 24. VH-VL of F6Aartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATATCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTATCCTTCTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 25. VH-VL-P of F6A artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 26. VH-VL-P ofF6A artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATATCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTATCCTTCTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 27. CDR-L1 of H2C artificial aaGSSTGAVTSGYYPN 28. CDR-L2 of H2C artificial aa GTKFLAP 29. CDR-L3 of H2Cartificial aa ALWYSNRWV 30. CDR-H1 of H2C artificial aa KYAMN 31. CDR-H2of H2C artificial aa RIRSKYNNYATYYADSVKD 32. CDR-H3 of H2C artificial aaHGNFGNSYISYWAY 33. VH of H2C artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 34. VH ofH2C artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 35. VL of H2Cartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 36. VL of H2C artificial ntCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 37.VH-P of H2C artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 38. VH-P ofH2C artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 39. VL-P of H2Cartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 40. VL-P of H2C artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 41.VH-VL of H2C artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 42. VH-VL of H2Cartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 43. VH-VL-P of H2C artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 44. VH-VL-P ofH2C artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 45. CDR-L1 of H1E artificial aaGSSTGAVTSGYYPN 46. CDR-L2 of H1E artificial aa GTKFLAP 47. CDR-L3 of H1Eartificial aa ALWYSNRWV 48. CDR-H1 of H1E artificial aa SYAMN 49. CDR-H2of H1E artificial aa RIRSKYNNYATYYADSVKG 50. CDR-H3 of H1E artificial aaHGNFGNSYLSFWAY 51. VH of H1E artificial aaEVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSS 52. VH ofH1E artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGAGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATTCGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACCTATCCTTCTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTC 53. VL of H1Eartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 54. VL of H1E artificial ntCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 55.VH-P of H1E artificial aaEVQLLESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSS 56. VH-P ofH1E artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGAGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATTCGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACCTATCCTTCTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 57. VL-P of H1Eartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 58. VL-P of H1E artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 59.VH-VL of H1E artificial aaEVQLVESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 60. VH-VL of H1Eartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGAGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATTCGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACCTATCCTTCTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 61. VH-VL-P of H1E artificial aaEVQLLESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 62. VH-VL-P ofH1E artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGAGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATTCGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACCTATCCTTCTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 63. CDR-L1 of G4H artificial aaGSSTGAVTSGYYPN 64. CDR-L2 of G4H artificial aa GTKFLAP 65. CDR-L3 of G4Hartificial aa ALWYSNRWV 66. CDR-H1 of G4H artificial aa RYAMN 67. CDR-H2of G4H artificial aa RIRSKYNNYATYYADSVKG 68. CDR-H3 of G4H artificial aaHGNFGNSYLSYFAY 69. VH of G4H artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSS 70. VH ofG4H artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATCGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTACTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 71. VL of G4Hartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 72. VL of G4H artificial ntCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 73.VH-P of G4H artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSS 74. VH-P ofG4H artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATCGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTACTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 75. VL-P of G4Hartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 76. VL-P of G4H artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 77.VH-VL of G4H artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 78. VH-VL of G4Hartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATCGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTACTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 79. VH-VL-P of G4H artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 80. VH-VL-P ofG4H artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATCGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTACTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 81. CDR-L1 of A2J artificial aaRSSTGAVTSGYYPN 82. CDR-L2 of A2J artificial aa ATDMRPS 83. CDR-L3 of A2Jartificial aa ALWYSNRWV 84. CDR-H1 of A2J artificial aa VYAMN 85. CDR-H2of A2J artificial aa RIRSKYNNYATYYADSVKK 86. CDR-H3 of A2J artificial aaHGNFGNSYLSWWAY 87. VH of A2J artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSS 88. VH ofA2J artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 89. VL of A2Jartificial aaQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 90. VL of A2J artificial ntCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 91.VH-P of A2J artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSS 92. VH-P ofA2J artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 93. VL-P of A2Jartificial aaELVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 94. VL-P of A2J artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA 95.VH-VL of A2J artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 96. VH-VL of A2Jartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 97. VH-VL-P of A2J artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 98. VH-VL-P ofA2J artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 99. CDR-L1 of E1L artificial aaGSSTGAVTSGYYPN 100. CDR-L2 of E1L artificial aa GTKFLAP 101. CDR-L3 ofE1L artificial aa ALWYSNRWV 102. CDR-H1 of E1L artificial aa KYAMN 103.CDR-H2 of E1L artificial aa RIRSKYNNYATYYADSVKS 104. CDR-H3 of E1Lartificial aa HGNFGNSYTSYYAY 105. VH of E1L artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSS 106. VH ofE1L artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAATCGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACACATCCTACTACGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 107. VL of E1Lartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 108. VL of E1L artificialnt CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA109. VH-P of E1L artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSS 110. VH-Pof E1L artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAATCGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACACATCCTACTACGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 111. VL-P of E1Lartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 112. VL-P of E1L artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA113. VH-VL of E1L artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 114. VH-VL of E1Lartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAATCGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACACATCCTACTACGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 115. VH-VL-P of E1L artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 116. VH-VL-P ofE1L artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAATCGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACACATCCTACTACGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 117. CDR-L1 of E2M artificial aaRSSTGAVTSGYYPN 118. CDR-L2 of E2M artificial aa ATDMRPS 119. CDR-L3 ofE2M artificial aa ALWYSNRWV 120. CDR-H1 of E2M artificial aa GYAMN 121.CDR-H2 of E2M artificial aa RIRSKYNNYATYYADSVKE 122. CDR-H3 of E2Martificial aa HRNFGNSYLSWFAY 123. VH of E2M artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSS 124. VH ofE2M artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATAGGAACTTCGGTAATAGCTACTTATCCTGGTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 125. VL of E2Martificial aaQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 126. VL of E2M artificialnt CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA127. VH-P of E2M artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSS 128. VH-Pof E2M artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATAGGAACTTCGGTAATAGCTACTTATCCTGGTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 129. VL-P of E2Martificial aaELVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 130. VL-P of E2M artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA131. VH-VL of E2M artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 132. VH-VL of E2Martificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATAGGAACTTCGGTAATAGCTACTTATCCTGGTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 133. VH-VL-P of E2M artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 134. VH-VL-P ofE2M artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATAGGAACTTCGGTAATAGCTACTTATCCTGGTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 135. CDR-L1 of F7O artificial aaGSSTGAVTSGYYPN 136. CDR-L2 of F7O artificial aa GTKFLAP 137. CDR-L3 ofF7O artificial aa ALWYSNRWV 138. CDR-H1 of F7O artificial aa VYAMN 139.CDR-H2 of F7O artificial aa RIRSKYNNYATYYADSVKK 140. CDR-H3 of F7Oartificial aa HGNFGNSYISWWAY 141. VH of F7O artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSS 142. VH ofF7O artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 143. VL of F7Oartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 144. VL of F7O artificialnt CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA145. VH-P of F7O artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSS 146. VH-Pof F7O artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 147. VL-P of F7Oartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 148. VL-P of F7O artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA149. VH-VL of F7O artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 150. VH-VL of F7Oartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 151. VH-VL-P of F7O artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 152. VH-VL-P ofF7O artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 153. CDR-L1 of F12Q artificial aaGSSTGAVTSGNYPN 154. CDR-L2 of F12Q artificial aa GTKFLAP 155. CDR-L3 ofF12Q artificial aa VLWYSNRWV 156. CDR-H1 of F12Q artificial aa SYAMN157. CDR-H2 of F12Q artificial aa RIRSKYNNYATYYADSVKG 158. CDR-H3 ofF12Q artificial aa HGNFGNSYVSWWAY 159. VH of F12Q artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS 160. VH ofF12Q artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 161. VL of F12Qartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 162. VL of F12Q artificialnt CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA163. VH-P of F12Q artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSS 164. VH-Pof F12Q artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 165. VL-P of F12Qartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 166. VL-P of F12Qartificial ntGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA167. VH-VL of F12Q artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 168. VH-VL ofF12Q artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 169. VH-VL-P of artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS F12QVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 170. VH-VL-P ofartificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATG F12QTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 171. CDR-L1 of I2C artificial aaGSSTGAVTSGNYPN 172. CDR-L2 of I2C artificial aa GTKFLAP 173. CDR-L3 ofI2C artificial aa VLWYSNRWV 174. CDR-H1 of I2C artificial aa KYAMN 175.CDR-H2 of I2C artificial aa RIRSKYNNYATYYADSVKD 176. CDR-H3 of I2Cartificial aa HGNFGNSYISYWAY 177. VH of I2C artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 178. VH ofI2C artificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 179. VL of I2Cartificial aaQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 180. VL of I2C artificialnt CAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA181. VH-P of I2C artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSS 182. VH-Pof I2C artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCA 183. VL-P of I2Cartificial aaELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 184. VL-P of I2C artificialnt GAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA185. VH-VL of I2C artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 186. VH-VL of I2Cartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 187. VH-VL-P of I2C artificial aaEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 188. VH-VL-P ofI2C artificial ntGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 189. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTETNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ VH-VLx GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG H2CVH-VL GSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 190. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG VH-VLx CAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG H2CVH-VL GTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGATATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA191. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ VH-VLx GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG F12QVH-VL GSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 192. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG VH-VLx CAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG F12QVH-VL GTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGATATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA193. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ VH-VLx GRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG I2CVH-VL GSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 194. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG VH-VLx CAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG I2CVH-VL GTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGATATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA195. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG F6AVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNIYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSFFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 196. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG F6AVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATATCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTATCCTTCTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA197. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG H2CVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 198. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG H2CVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA199. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG H1EVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLEQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSFWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 200. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG H1EVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGAGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATTCGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACCTATCCTTCTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA201. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG G4HVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 202. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG G4HVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATCGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTACTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA203. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG A2JVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 204. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG A2JVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA205. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG E1LVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKSRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYTSYYAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 206. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG E1LVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAATCGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACACATCCTACTACGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA207. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG E2MVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNGYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKERFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHRNFGNSYLSWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCRSSTGAVTSGYYPNWVQQKPGQAPRGLIGATDMRPSGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 208. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG E2MVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATAGGAACTTCGGTAATAGCTACTTATCCTGGTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTCGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGCCACTGACATGAGGCCCTCTGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA209. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG F7OVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNVYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKKRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 210. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG F7OVH-VL-PGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATGTGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAAAGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA211. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG F12QVH-VL GSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 212. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG F12QVH-VL GTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA213. MCSP-G4 artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG I2CVH-VL GSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 214. MCSP-G4 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAG I2CVH-VL GTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA215. MCSP-D2 artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTSYAQKFQ VH-VLx GRVTMTRDTSTSTVYMELSNLRSDDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG H2CVH-VL GSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 216. MCSP-D2 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG VH-VLx CAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAG H2CVH-VL GGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACTAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAACCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGATATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA217. MCSP-D2 artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTSYAQKFQ VH-VLx GRVTMTRDTSTSTVYMELSNLRSDDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG F12QVH-VL GSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 218. MCSP-D2 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG VH-VLx CAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAG F12QVH-VL GGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACTAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAACCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGATATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA219. MCSP-D2 artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTSYAQKFQ VH-VLx GRVTMTRDTSTSTVYMELSNLRSDDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG I2CVH-VL GSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 220. MCSP-D2 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG VH-VLx CAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAG I2CVH-VL GGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACTAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAACCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGATATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA221. MCSP-D2 artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTSYAQKFQVH-VL-P xGRVTMTRDTSTSTVYMELSNLRSDDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG H2CVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLNSSNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 222. MCSP-D2 artificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGVH-VL-P xCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAG H2CVH-VL-PGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAGCTACGCACAGAAGTTCCAGGGCAGAGTCACCATGACTAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAACCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCTCCAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA223. MCSP-F9 artificial aaQVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWLGTIYYNGNTYYNPSLK VH-VLx SRVTISVDTSKNQFSLRLSSVTAADTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG H2CVH-VL GSDIVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSVPFTFGPGTKVDIKGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 224. MCSP-F9 artificial ntCAGGTGCAGCTGCAAGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTG VH-VLx CGTTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGCTGGGTCCGCCAGCCCCCAGGGA H2CVH-VL AGGGACTGGAGTGGCTTGGGACTATCTATTATAATGGGAATACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATCTCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGATATCGTGATGACACAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTCTTATCCAGCTCCAACAATAAGAACTACTTAAATTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTGTTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCC TA225. MCSP-F9 artificial aaEVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWLGTIYYNGNTYYNPSLKVH-VL-P xSRVTISVDTSKNQFSLRLSSVTAADTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG H2CVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSVPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 226. MCSP-F9 artificial ntGAGGTGCAGCTGCAAGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGVH-VL-P xCGTTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGCTGGGTCCGCCAGCCCCCAGGGA H2CVH-VL-PAGGGACTGGAGTGGCTTGGGACTATCTATTATAATGGGAATACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATCTCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACACAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTCTTATCCAGCTCCAACAATAAGAACTACTTAAATTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTGTTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA227. MCSP-F9 artificial aaEVQLQESGPGLVKPSETLSLTCVVSGGSISSSNWWSWVRQPPGKGLEWLGTIYYNGNTYYNPSLKVH-VL-P xSRVTISVDTSKNQFSLRLSSVTAADTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGG G4HVH-VL-PGSELVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSVPFTFGPGTKVDIKSGGGGSEVQLLESGGGLVQPGGSLKLSCAASGFTFNRYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYLSYFAYWGQGTLVTVSSGGGGSGGGGSGGGGSELVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 228. MCSP-F9 artificial ntGAGGTGCAGCTGCAAGAGTCTGGCCCAGGACTGGTGAAGCCTTCGGAGACCCTGTCCCTCACCTGVH-VL-P xCGTTGTCTCTGGTGGCTCCATCAGCAGTAGTAACTGGTGGAGCTGGGTCCGCCAGCCCCCAGGGA G4HVH-VL-PAGGGACTGGAGTGGCTTGGGACTATCTATTATAATGGGAATACCTACTACAACCCGTCCCTCAAGAGTCGAGTCACCATCTCCGTAGACACGTCCAAGAACCAGTTCTCCCTGAGGCTGAGCTCTGTGACCGCCGCAGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTGATGACACAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTCTTATCCAGCTCCAACAATAAGAACTACTTAAATTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTGTTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGCTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATCGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGGAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACTTATCCTACTTCGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGAGCTCGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA229. 1-27 CD3ε-Fc artificial aaQDGNEEMGGITQTPYKVSISGTTVILTSGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH 230. 1-27 CD3ε-Fc artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCAAGATGGTAATGAAGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCACAGTAATATTGACATCCGGAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAACATCATCACCATCATCAT 231. human 1-27 artificial aaQDGNEEMGGITQTPYKVSISGTTVILTDYKDDDDKTASFAAAQKECVCENYKLAVNCFLNDNGQC CD3ε -EpCAM QCTSIGAQNTVLCSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSTCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDVQSLRTALEEAIKTRYQLDPKFITNILYEDNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLRVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 232. human 1-27 artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCAAGATGG CD3ε -EpCAM TAATGAAGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCACAGTAATATTGACAGATTACAAGGACGACGATGACAAGACTGCGAGTTTTGCCGCAGCTCAGAAAGAATGTGTCTGTGAAAACTACAAGCTGGCCGTAAACTGCTTTTTGAATGACAATGGTCAATGCCAGTGTACTTCGATTGGTGCACAAAATACTGTCCTTTGCTCAAAGCTGGCTGCCAAATGTTTGGTGATGAAGGCAGAAATGAACGGCTCAAAACTTGGGAGAAGAGCGAAACCTGAAGGGGCTCTCCAGAACAATGATGGCCTTTACGATCCTGACTGCGATGAGAGCGGGCTCTTTAAGGCCAAGCAGTGCAACGGCACCTCCACGTGCTGGTGTGTGAACACTGCTGGGGTCAGAAGAACTGACAAGGACACTGAAATAACCTGCTCTGAGCGAGTGAGAACCTACTGGATCATCATTGAATTAAAACACAAAGCAAGAGAAAAACCTTATGATGTTCAAAGTTTGCGGACTGCACTTGAGGAGGCGATCAAAACGCGTTATCAACTGGATCCAAAATTTATCACAAATATTTTGTATGAGGATAATGTTATCACTATTGATCTGGTTCAAAATTCTTCTCAGAAAACTCAGAATGATGTGGACATAGCTGATGTGGCTTATTATTTTGAAAAAGATGTTAAAGGTGAATCCTTGTTTCATTCTAAGAAAATGGACCTGAGAGTAAATGGGGAACAACTGGATCTGGATCCTGGTCAAACTTTAATTTATTATGTCGATGAAAAAGCACCTGAATTCTCAATGCAGGGTCTAAAAGCTGGTGTTATTGCTGTTATTGTGGTTGTGGTGATAGCAATTGTTGCTGGAATTGTTGTGCTGGTTATTTCCAGAAAGAAGAGAATGGCAAAGTATGAGAAGGCTGAGATAAAGGAGATGGGTGAGATGCATAGGGAACTCAATGCA 233. marmoset 1-27 artificial aaQDGNEEMGDTTQNPYKVSISGTTVTLTDYKDDDDKTASFAAAQKECVCENYKLAVNCFLNDNGQC CD3ε-EpCAM QCTSIGAQNTVLCSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSTCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDVQSLRTALEEAIKTRYQLDPKFITNILYEDNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLRVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 234. marmoset 1-27 artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCAGGACGG CD3ε-EpCAM TAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACAGATTACAAGGACGACGATGACAAGACTGCGAGTTTTGCCGCAGCTCAGAAAGAATGTGTCTGTGAAAACTACAAGCTGGCCGTAAACTGCTTTTTGAATGACAATGGTCAATGCCAGTGTACTTCGATTGGTGCACAAAATACTGTCCTTTGCTCAAAGCTGGCTGCCAAATGTTTGGTGATGAAGGCAGAAATGAACGGCTCAAAACTTGGGAGAAGAGCGAAACCTGAAGGGGCTCTCCAGAACAATGATGGCCTTTACGATCCTGACTGCGATGAGAGCGGGCTCTTTAAGGCCAAGCAGTGCAACGGCACCTCCACGTGCTGGTGTGTGAACACTGCTGGGGTCAGAAGAACTGACAAGGACACTGAAATAACCTGCTCTGAGCGAGTGAGAACCTACTGGATCATCATTGAATTAAAACACAAAGCAAGAGAAAAACCTTATGATGTTCAAAGTTTGCGGACTGCACTTGAGGAGGCGATCAAAACGCGTTATCAACTGGATCCAAAATTTATCACAAATATTTTGTATGAGGATAATGTTATCACTATTGATCTGGTTCAAAATTCTTCTCAGAAAACTCAGAATGATGTGGACATAGCTGATGTGGCTTATTATTTTGAAAAAGATGTTAAAGGTGAATCCTTGTTTCATTCTAAGAAAATGGACCTGAGAGTAAATGGGGAACAACTGGATCTGGATCCTGGTCAAACTTTAATTTATTATGTCGATGAAAAAGCACCTGAATTCTCAATGCAGGGTCTAAAAGCTGGTGTTATTGCTGTTATTGTGGTTGTGGTGATAGCAATTGTTGCTGGAATTGTTGTGCTGGTTATTTCCAGAAAGAAGAGAATGGCAAAGTATGAGAAGGCTGAGATAAAGGAGATGGGTGAGATGCATAGGGAACTCAATGCA 235. tamarin 1-27 artificial aaQDGNEEMGDTTQNPYKVSISGTTVTLTDYKDDDDKTASFAAAQKECVCENYKLAVNCFLNDNGQC CD3ε -EpCAM QCTSIGAQNTVLCSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSTCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDVQSLRTALEEAIKTRYQLDPKFITNILYEDNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLRVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 236. tamarin 1-27 artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCAGGACGG CD3ε -EpCAM TAATGAAGAAATGGGTGATACTACACAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACACTGACAGATTACAAGGACGACGATGACAAGACTGCGAGTTTTGCCGCAGCTCAGAAAGAATGTGTCTGTGAAAACTACAAGCTGGCCGTAAACTGCTTTTTGAATGACAATGGTCAATGCCAGTGTACTTCGATTGGTGCACAAAATACTGTCCTTTGCTCAAAGCTGGCTGCCAAATGTTTGGTGATGAAGGCAGAAATGAACGGCTCAAAACTTGGGAGAAGAGCGAAACCTGAAGGGGCTCTCCAGAACAATGATGGCCTTTACGATCCTGACTGCGATGAGAGCGGGCTCTTTAAGGCCAAGCAGTGCAACGGCACCTCCACGTGCTGGTGTGTGAACACTGCTGGGGTCAGAAGAACTGACAAGGACACTGAAATAACCTGCTCTGAGCGAGTGAGAACCTACTGGATCATCATTGAATTAAAACACAAAGCAAGAGAAAAACCTTATGATGTTCAAAGTTTGCGGACTGCACTTGAGGAGGCGATCAAAACGCGTTATCAACTGGATCCAAAATTTATCACAAATATTTTGTATGAGGATAATGTTATCACTATTGATCTGGTTCAAAATTCTTCTCAGAAAACTCAGAATGATGTGGACATAGCTGATGTGGCTTATTATTTTGAAAAAGATGTTAAAGGTGAATCCTTGTTTCATTCTAAGAAAATGGACCTGAGAGTAAATGGGGAACAACTGGATCTGGATCCTGGTCAAACTTTAATTTATTATGTCGATGAAAAAGCACCTGAATTCTCAATGCAGGGTCTAAAAGCTGGTGTTATTGCTGTTATTGTGGTTGTGGTGATAGCAATTGTTGCTGGAATTGTTGTGCTGGTTATTTCCAGAAAGAAGAGAATGGCAAAGTATGAGAAGGCTGAGATAAAGGAGATGGGTGAGATGCATAGGGAACTCAATGCA 237. squirrel artificial aaQDGNEEIGDTTQNPYKVSISGTTVTLTDYKDDDDKTASFAAAQKECVCENYKLAVNCFLNDNGQC monkey1-27 QCTSIGAQNTVLCSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNCD3ε -EpCAMGTSTCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDVQSLRTALEEAIKTRYQLDPKFITNILYEDNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLRVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 238. squirrel artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCAGGACGG monkey1-27 TAATGAAGAGATTGGTGATACTACCCAGAACCCATATAAAGTTTCCATCTCAGGAACCACAGTAACD3ε -EpCAMCACTGACAGATTACAAGGACGACGATGACAAGACTGCGAGTTTTGCCGCAGCTCAGAAAGAATGTGTCTGTGAAAACTACAAGCTGGCCGTAAACTGCTTTTTGAATGACAATGGTCAATGCCAGTGTACTTCGATTGGTGCACAAAATACTGTCCTTTGCTCAAAGCTGGCTGCCAAATGTTTGGTGATGAAGGCAGAAATGAACGGCTCAAAACTTGGGAGAAGAGCGAAACCTGAAGGGGCTCTCCAGAACAATGATGGCCTTTACGATCCTGACTGCGATGAGAGCGGGCTCTTTAAGGCCAAGCAGTGCAACGGCACCTCCACGTGCTGGTGTGTGAACACTGCTGGGGTCAGAAGAACTGACAAGGACACTGAAATAACCTGCTCTGAGCGAGTGAGAACCTACTGGATCATCATTGAATTAAAACACAAAGCAAGAGAAAAACCTTATGATGTTCAAAGTTTGCGGACTGCACTTGAGGAGGCGATCAAAACGCGTTATCAACTGGATCCAAAATTTATCACAAATATTTTGTATGAGGATAATGTTATCACTATTGATCTGGTTCAAAATTCTTCTCAGAAAACTCAGAATGATGTGGACATAGCTGATGTGGCTTATTATTTTGAAAAAGATGTTAAAGGTGAATCCTTGTTTCATTCTAAGAAAATGGACCTGAGAGTAAATGGGGAACAACTGGATCTGGATCCTGGTCAAACTTTAATTTATTATGTCGATGAAAAAGCACCTGAATTCTCAATGCAGGGTCTAAAAGCTGGTGTTATTGCTGTTATTGTGGTTGTGGTGATAGCAATTGTTGCTGGAATTGTTGTGCTGGTTATTTCCAGAAAGAAGAGAATGGCAAAGTATGAGAAGGCTGAGATAAAGGAGATGGGTGAGATGCATAGGGAACTCAATGCA 239. swine 1-27 artificial aaQEDIERPDEDTQKTFKVSISGDKVELTDYKDDDDKTASFAAAQKECVCENYKLAVNCFLNDNGQC CD3ε -EpCAM QCTSIGAQNTVLCSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSTCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDVQSLRTALEEAIKTRYQLDPKFITNILYEDNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLRVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAIVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA 240. swine 1-27 artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCAAGAAGA CD3ε -EpCAM CATTGAAAGACCAGATGAAGATACACAGAAAACATTTAAAGTCTCCATCTCTGGAGACAAAGTAGAGCTGACAGATTACAAGGACGACGATGACAAGACTGCGAGTTTTGCCGCAGCTCAGAAAGAATGTGTCTGTGAAAACTACAAGCTGGCCGTAAACTGCTTTTTGAATGACAATGGTCAATGCCAGTGTACTTCGATTGGTGCACAAAATACTGTCCTTTGCTCAAAGCTGGCTGCCAAATGTTTGGTGATCAAGGCAGAAATGAACGGCTCAAAACTTGGGAGAAGAGCGAAACCTGAAGGGGCTCTCCAGAACAATGATGGCCTTTACGATCCTGACTGCGATGAGAGCGGGCTCTTTAAGGCCAAGCAGTGCAACGGCACCTCCACGTGCTGGTGTGTGAACACTGCTGGGGTCAGAAGAACTGACAAGGACACTGAAATAACCTGCTCTGAGCGAGTGAGAACCTACTGGATCATCATTGAATTAAAACACAAAGCAAGAGAAAAACCTTATGATGTTCAAAGTTTGCGGACTGCACTTGAGGAGGCGATCAAAACGCGTTATCAACTGGATCCAAAATTTATCACAAATATTTTGTATGAGGATAATGTTATCACTATTGATCTGGTTCAAAATTCTTCTCAGAAAACTCAGAATGATGTGGACATAGCTGATGTGGCTTATTATTTTGAAAAAGATGTTAAAGGTGAATCCTTGTTTCATTCTAAGAAAATGGACCTGAGAGTAAATGGGGAACAACTGGATCTGGATCCTGGTCAAACTTTAATTTATTATGTCGATGAAAAAGCACCTGAATTCTCAATGCAGGGTCTAAAAGCTGGTGTTATTGCTGTTATTGTGGTTGTGGTGATAGCAATTGTTGCTGGAATTGTTGTGCTGGTTATTTCCAGAAAGAAGAGAATGGCAAAGTATGAGAAGGCTGAGATAAAGGAGATGGGTGAGATGCATAGGGAACTCAATGCA 241. human CD3 human aaQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEepsilonFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYW chainSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI 242. human CD3human ntATGCAGTCGGGCACTCACTGGAGAGTTCTGGGCCTCTGCCTCTTATCAGTTGGCGTTTGGGGGCAepsilonAGATGGTAATGAAGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCA chainCAGTAATATTGACATGCCCTCAGTATCCTGGATCTGAAATACTATGGCAACACAATGATAAAAACATAGGCGGTGATGAGGATGATAAAAACATAGGCAGTGATGAGGATCACCTGTCACTGAAGGAATTTTCAGAATTGGAGCAAAGTGGTTATTATGTCTGCTACCCCAGAGGAAGCAAACCAGAAGATGCGAACTTTTATCTCTACCTGAGGGCACGCGTGTGTGAGAACTGCATGGAGATGGATGTGATGTCGGTGGCCACAATTGTCATAGTGGACATCTGCATCACTGGGGGCTTGCTGCTGCTGGTTTACTACTGGAGCAAGAATAGAAAGGCCAAGGCCAAGCCTGTGACACGAGGAGCGGGTGCTGGCGGCAGGCAAAGGGGACAAAACAAGGAGAGGCCACCACCTGTTCCCAACCCAGACTATGAGCCCATCCGGAAAGGCCAGCGGGACCTGTATTCTGGCCTGAATCAGAGACGCATC 243. 19 amino acid artificial aaMGWSCIILFLVATATGVHS immunoglobulin leader peptide 244. 19 amino acidartificial nt ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCimmunoglobulin leader peptide 245. murine IgG1 murine aaAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLS heavySSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTI chainTLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEconstantFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQW regionNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK 246.murine IgG1 murine ntGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCAT heavyGGTGACCCTGGGATGCCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTG chainGATCCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTACACTCTGAGCconstantAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCGTCACCTGCAACGTTGCCCACCC regionGGCCAGCAGCACCAAGGTGGACAAGAAAATTGTGCCCAGGGATTGTGGTTGTAAGCCTTGCATATGTACAGTCCCAGAAGTATCATCTGTCTTCATCTTCCCCCCAAAGCCCAAGGATGTGCTCACCATTACTCTGACTCCTAAGGTCACGTGTGTTGTGGTAGACATCAGCAAGGATGATCCCGAGGTCCAGTTCAGCTGGTTTGTAGATGATGTGGAGGTGCACACAGCTCAGACGCAACCCCGGGAGGAGCAGTTCAACAGCACTTTCCGCTCAGTCAGTGAACTTCCCATCATGCACCAGGACTGGCTCAATGGCAAGGAGTTCAAATGCAGGGTCAACAGTGCAGCTTTCCCTGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGCAGACCGAAGGCTCCACAGGTGTACACCATTCCACCTCCCAAGGAGCAGATGGCCAAGGATAAAGTCAGTCTGACCTGCATGATAACAGACTTCTTCCCTGAAGACATTACTGTGGAGTGGCAGTGGAATGGGCAGCCAGCGGAGAACTACAAGAACACTCAGCCCATCATGGACACAGATGGCTCTTACTTCGTCTACAGCAAGCTCAATGTGCAGAAGAGCAACTGGGAGGCAGGAAATACTTTCACCTGCTCTGTGTTACATGAGGGCCTGCACAACCACCATACTGAGAAGAGCCTCTCCCACTCTCCTGGTAAA 247.human lambda human aaGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNK lightYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS chain constant region 248.human lambda human ntGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCCTCTGAGGAGCTTCAAGCCAA lightCAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGG chainCAGATAGCAGCCCCGTCAAGGCGGGAGTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGconstantTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTG regionCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA 249.c-terminal human aaDYKDDDDKSRTRSGSQLDGGLVLFSHRGTLDGGFRFRLSDGEHTSPGHFFRVTAQKQVLLSLKGS domainQTLTVCPGSVQPLSSQTLRASSSAGTDPQLLLYRVVRGPQLGRLFHAQQDSTGEALVNFTQAEVYconstruct ofAGNILYEHEMPPEPFWEAHDTLELQLSSPPARDVAATLAVAVSFEAACPQRPSHLWKNKGLWVPE humanGQRARITVAALDASNLLASVPSPQRSEHDVLFQVTQFPSRGQLLVSEEPLHAGQPHFLQSQLAAG MCSPQLVYAHGGGGTQQDGFHFRAHLQGPAGASVAGPQTSEAFAITVRDVNERPPQPQASVPLRLTRGSRAPISRAQLSVVDPDSAPGEIEYEVQRAPHNGFLSLVGGGLGPVTRFTQADVDSGRLAFVANGSSVAGIFQLSMSDGASPPLPMSLAVDILPSAIEVQLRAPLEVPQALGRSSLSQQQLRVVSDRFEPEAAYRLIQGPQYGHLLVGGRPTSAFSQFQIDQGEVVFAFTNSSSSHDHFRVLALARGVNASAVVNVTVRALLHVWAGGPWPQGATLRLDPTVLDAGELANRTDSVPRFRLLEGPRHGRVVRVPRARTEPGGSQLVEQFTQQDLEDGRLGLEVGRPEGRAPGPAGDSLTLELWAQGVPPAVASLDFATEPYNAARPYSVALLSVPEAARTEAGKPESSTPTGEPGPMASSPEPAVAKGGFLSFLEANMFSVIIPMCLVLLLLALILPLLFYLRKRNKTGKHDVQVLTAKPRNGLAGDTETFRKVEPGQAIPLTAVPGQGPPPGGQPDPELLQFCRTPNPALKNGQYWV 250. c-terminal human ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCGACTACAA domainAGACGATGACGACAAGTCCCGTACGAGATCTGGATCCCAATTGGACGGCGGGCTCGTGCTGTTCTconstruct ofCACACAGAGGAACCCTGGATGGAGGCTTCCGCTTCCGCCTCTCTGACGGCGAGCACACTTCCCCC humanGGACACTTCTTCCGAGTGACGGCCCAGAAGCAAGTGCTCCTCTCGCTGAAGGGCAGCCAGACACT MCSPGACTGTCTGCCCAGGGTCCGTCCAGCCACTCAGCAGTCAGACCCTCAGGGCCAGCTCCAGCGCAGGCACTGACCCCCAGCTCCTGCTCTACCGTGTGGTGCGGGGCCCCCAGCTAGGCCGGCTGTTCCACGCCCAGCAGGACAGCACAGGGGAGGCCCTGGTGAACTTCACTCAGGCAGAGGTCTACGCTGGGAATATTCTGTATGAGCATGAGATGCCCCCCGAGCCCTTTTGGGAGGCCCATGATACCCTAGAGCTCCAGCTGTCCTCGCCGCCTGCCCGGGACGTGGCCGCCACCCTTGCTGTGGCTGTGTCTTTTGAGGCTGCCTGTCCCCAGCGCCCCAGCCACCTCTGGAAGAACAAAGGTCTCTGGGTCCCCGAGGGCCAGCGGGCCAGGATCACCGTGGCTGCTCTGGATGCCTCCAATCTCTTGGCCAGCGTTCCATCACCCCAGCGCTCAGAGCATGATGTGCTCTTCCAGGTCACACAGTTCCCCAGCCGCGGCCAGCTGTTGGTGTCCGAGGAGCCCCTCCATGCTGGGCAGCCCCACTTCCTGCAGTCCCAGCTGGCTGCAGGGCAGCTAGTGTATGCCCACGGCGGTGGGGGCACCCAGCAGGATGGCTTCCACTTTCGTGCCCACCTCCAGGGGCCAGCAGGGGCCTCCGTGGCTGGACCCCAAACCTCAGAGGCCTTTGCCATCACGGTGAGGGATGTAAATGAGCGGCCCCCTCAGCCACAGGCCTCTGTCCCACTCCGGCTCACCCGAGGCTCTCGTGCCCCCATCTCCCGGGCCCAGCTGAGTGTGGTGGACCCAGACTCAGCTCCTGGGGAGATTGAGTACGAGGTCCAGCGGGCACCCCACAACGGCTTCCTCAGCCTGGTGGGTGGTGGCCTGGGGCCCGTGACCCGCTTCACGCAAGCCGATGTGGATTCAGGGCGGCTGGCCTTCGTGGCCAACGGGAGCAGCGTGGCAGGCATCTTCCAGCTGAGCATGTCTGATGGGGCCAGCCCACCCCTGCCCATGTCCCTGGCTGTGGACATCCTACCATCCGCCATCGAGGTGCAGCTGCGGGCACCCCTGGAGGTGCCCCAAGCTTTGGGGCGCTCCTCACTGAGCCAGCAGCAGCTCCGGGTGGTTTCAGATCGGGAGGAGCCAGAGGCAGCATACCGGTTGATCCAGGGACCCCAGTATGGGCATCTCCTGGTGGGCGGGCGGCCCACCTCGGCCTTCAGCCAATTCCAGATAGACCAGGGCGAGGTGGTCTTTGCCTTCACCAACTCCTCCTCCTCTCATGACCACTTCAGAGTCCTGGCACTGGCTAGGGGTGTCAATGCATCAGCCGTAGTGAACGTCACTGTGAGGGCTCTGCTGCATGTGTGGGCAGGTGGGCCATGGCCCCAGGGTGCCACCCTGCGCCTGGACCCCACCGTCCTAGATGCTGGCGAGCTGGCCAACCGCACAGACAGTGTGCCGCGCTTCCGCCTCCTGGAGGGACCCCGGCATGGCCGCGTGGTCCGCGTGCCCCGAGCCAGGACGGAGCCCGGGGGCAGCCAGCTGGTGGAGCAGTTCACTCAGCAGGACCTTGAGGACGGGAGGCTGGGGCTGGAGGTGGGCAGGCCAGAGGGGAGGGCCCCCGGCCCCGCAGGTGACAGTCTCACTCTGGAGCTGTGGGCACAGGGCGTCCCGCCTGCTGTGGCCTCCCTGGACTTTGCCACTGAGCCTTACAATGCTGCCCGGCCCTACAGCGTGGCCCTGCTCAGTGTCCCCGAGGCCGCCCGGACGGAAGCAGGGAAGCCAGAGAGCAGCACCCCCACAGGCGAGCCAGGCCCCATGGCATCCAGCCCTGAGCCCGCTGTGGCCAAGGGAGGCTTCCTGAGCTTTCTAGAGGCCAACATGTTCAGCGTCATCATCCCCATGTGCCTGGTACTTCTGCTCCTGGCGCTCATCCTGCCCCTGCTCTTCTACCTCCGAAAACGCAACAAGACGGGCAAGCATGACGTCCAGGTCCTGACTGCCAAGCCCCGCAACGGCCTGGCTGGTGACACCGAGACCTTTCGCAAGGTGGAGCCAGGCCAGGCCATCCCGCTCACAGCTGTGCCTGGCCAGGGGCCCCCTCCAGGAGGCCAGCCTGACCCAGAGCTGCTGCAGTTCTGCCGGACACCCAACCCTGCCCTTAAGAATGGCCAGTACTGGGTG 251. partialcynomolgus aaPSNGRVVLRAAPGTEVRSETQAQLDGGLVLFSHRGTLDGGFREGLSDGEHTSSGHFFRVTAQKQVsequence ofLLSLEGSRTLTVCPGSVQPLSSQTLRASSSAGTDPQLLLYRVVRGPQLGRLFHAQQDSTGEALVNcynomolgusFTQAEVYAGNILYEHEMPTEPFWEAHDTLELQLSSPPARDVAATLAVAVSFEAACPQRPSHLWKN MCSPKGLWVPEGQRAKITMAALDASNLLASVPSSQRLEHDVLFQVTQFPSRGQLLVSEEPLHAGQPHFLQSQLAAGQLVYAHGGGGTQQDGFHFRAHLQGPAGATVAGPQTSEAFAITVRDVNERPPQPQASVPLRITRGSRAPISRAQLSVVDPDSAPGEIEYEVQRAPHNGFLSLVGGGPGPVNRFTQADVDSGRLAFVANGSSVAGVFQLSMSDGASPPLPMSLAVDILPSAIEVQLQAPLEVPQALGRSSLSQQQLRVVSDREEPEAAYRLIQGPKYGHLLVGGQPASAFSQLQIDQGEVVFAFTNFSSSHDHFRVLALARGVNASAVVNITVRALLHVWAGGPWPQGATLRLDPTILDAGELANRTGSVPRFRLLEGPRHGRVVRVPRARMEPGGSQLVEQFTQQDLEDGRLGLEVGRPEGRAPSPTGDSLTLELWAQGVPPAVASLDFATEPYNAARPYSVALLSVPEATRTEAGKPESSTPTGEPGPMASSPVPAVAKGGFLGFLEANMFSVIIPXCLVLLLLALILPLLFYLRKRNKTGKHDVQVLTAKPRNGLAGDTETFRKVEPGQAIPLTAVPGQGPPPGGQPDPELLQFCRTPNPALKNGQYWV 252. partial cynomolgus ntCCCAGCAACGGACGGGTAGTGCTGCGGGCGGCGCCGGGCACCGAGGTGCGCAGCTTCACGCAGGCsequence ofCCAGCTGGATGGCGGACTCGTGCTGTTCTCACACAGAGGAACCCTGGATGGAGGCTTCCGCTTCGcynomolgusGCCTCTCCGATGGCGAGCACACTTCCTCTGGACACTTCTTCCGAGTGACGGCCCAGAAGCAAGTG MCSPCTCCTCTCGCTGGAGGGCAGCCGGACACTGACTGTCTGCCCAGGGTCCGTGCAGCCACTCAGCAGTCAGACCCTCAGAGCCAGCTCCAGCGCAGGCACCGACCCCCAGCTCCTGCTCTACCGTGTGGTGCGGGGCCCCCAGCTAGGCCGGCTGTTCCATGCCCAGCAGGACAGCACAGGGGAGGCCCTGGTGAACTTCACTCAGGCAGAGGTCTATGCTGGGAATATTCTGTATGAGCATGAGATGCCCACCGAGCCCTTCTGGGAGGCCCATGATACCCTAGAGCTCCAGCTGTCCTCACCACCTGCCCGGGACGTGGCTGCCACCCTTGCTGTGGCTGTGTCTTTTGAGGCTGCCTGTCCCCAGCGCCCCAGCCACCTCTGGAAGAACAAAGGTCTCTGGGTCCCCGAGGGCCAGCGGGCCAAGATCACCATGGCTGCCCTGGATGCCTCCAACCTCTTGGCCAGCGTTCCATCATCCCAGCGCCTAGAGCATGATGTGCTCTTCCAGGTCACGCAGTTCCCCAGCCGGGGCCAGCTATTGGTGTCTGAGGAGCCCCTCCACGCTGGGCAGCCCCACTTCCTGCAGTCCCAGCTGGCTGCAGGGCAGCTAGTGTATGCCCACGGCGGTGGGGGTACCCAACAGGATGGCTTCCACTTTCGTGCCCACCTCCAGGGGCCAGCAGGGGCCACCGTGGCTGGACCCCAAACCTCAGAGGCTTTTGCCATCACGGTGCGGGATGTAAATGAGCGGCCCCCTCAGCCACAGGCCTCTGTCCCACTCCGGATCACCCGAGGCTCTCGAGCCCCCATCTCCCGGGCCCAGCTGAGTGTCGTGGACCCAGACTCAGCTCCTGGGGAGATTGAGTATGAGGTCCAGCGGGCACCCCACAACGGCTTCCTCAGCCTGGTGGGTGGTGGCCCGGGGCCCGTGAACCGCTTCACGCAAGCCGATGTGGATTCGGGGCGGCTGGCCTTCGTGGCCAACGGGAGCAGCGTAGCAGGCGTCTTCCAGCTGAGCATGTCTGATGGGGCCAGCCCACCGCTGCCCATGTCCCTGGCCGTGGACATCCTACCATCCGCCATCGAGGTGCAGCTGCAGGCACCCCTGGAGGTGCCCCAAGCTTTGGGGCGCTCCTCACTGAGCCAGCAGCAGCTCCGGGTGGTTTCAGATAGGGAGGAGCCAGAGGCAGCATACCGCCTCATCCAGGGACCAAAGTACGGGCATCTCCTGGTGGGTGGGCAGCCCGCCTCGGCCTTCAGCCAACTCCAGATAGACCAGGGCGAGGTGGTCTTTGCCTTCACCAACTTCTCCTCCTCTCATGACCACTTCAGAGTCCTGGCACTGGCTAGGGGTGTCAACGCATCAGCCGTAGTGAACATCACTGTGAGGGCTCTGCTGCACGTGTGGGCAGGTGGGCCATGGCCCCAGGGTGCTACCCTGCGCCTGGACCCAACCATCCTAGATGCTGGCGAGCTGGCCAACCGCACAGGCAGTGTGCCCCGCTTCCGCCTCCTGGAGGGACCCCGGCATGGCCGCGTGGTCCGTGTGCCCCGAGCCAGGATGGAGCCTGGGGGCAGCCAGCTGGTGGAGCAGTTCACTCAGCAGGACCTTGAGGATGGGAGGCTGGGGCTGGAGGTGGGCAGGCCAGAGGGAAGGGCCCCCAGCCCCACAGGCGACAGTCTCACTCTGGAGCTGTGGGCACAGGGCGTCCCACCTGCTGTGGCCTCCCTGGACTTTGCCACTGAGCCTTACAATGCTGCCCGGCCCTACAGCGTGGCCCTGCTCAGTGTCCCCGAGGCCACCCGGACGGAAGCAGGGAAGCCAGAGAGCAGCACCCCCACAGGCGAGCCAGGCCCCATGGCATCTAGCCCTGTGCCTGCTGTGGCCAAGGGAGGCTTCCTGGGCTTCCTTGAGGCCAACATGTTCAGTGTCATCATCCCCRTGTGCCTGGTCCTTCTGCTCCTGGCGCTCATCTTGCCCCTGCTCTTCTACCTCCGAAAACGCAACAAGACGGGCAAGCATGACGTCCAGGTCCTGACTGCCAAGCCCCGCAATGGTCTGGCTGGTGACACTGAGACCTTTCGCAAGGTGGAGCCAGGCCAGGCCATCCCGCTCACAGCTGTGCCTGGCCAGGGGCCCCCTCCGGGAGGCCAGCCTGACCCAGAGCTGCTGCAGTTCTGCCGGACACCCAACCCTGCCCTTAAGAATGGCCAGTACTGGGTG 253. PCR primer for artificial nt AGAGTTCTGGGCCTCTGCCD3ε chain - forward primer 254. PCR primer artificial ntCGGATGGGCTCATAGTCTG for CD3ε chain - reverse primer 255. His6-humanartificial aaHHHHHHQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQHNDKNIGGDEDDKNIGSDED CD3εHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI 256.His6-human artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCATCATCA CD3εCCATCATCATCAAGATGGTAATGAAGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCACAGTAATATTGACATGCCCTCAGTATCCTGGATCTGAAATACTATGGCAACACAATGATAAAAACATAGGCGGTGATGAGGATGATAAAAACATAGGCAGTGATGAGGATCACCTGTCACTGAAGGAATTTTCAGAATTGGAGCAAAGTGGTTATTATGTCTGCTACCCCAGAGGAAGCAAACCAGAAGATGCGAACTTTTATCTCTACCTGAGGGCACGCGTGTGTGAGAACTGCATGGAGATGGATGTGATGTCGGTGGCCACAATTGTCATAGTGGACATCTGCATCACTGGGGGCTTGCTGCTGCTGGTTTACTACTGGAGCAAGAATAGAAAGGCCAAGGCCAAGCCTGTGACACGAGGAGCGGGTGCTGGCGGCAGGCAAAGGGGACAAAACAAGGAGAGGCCACCACCTGTTCCCAACCCAGACTATGAGCCCATCCGGAAAGGCCAGCGGGACCTGTATTCTGGCCTGAATCAGAGACGCATC 257. CD33 AH3 HL xartificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFK H2C HLGRVTMSSDTSTSTAYLEINSLRSDDTAIYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTENKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 258. CD33 AH3 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAAAAGCCTGGAGAGTCAGTCAAGGTCTCCTG H2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAGGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGTCTTCGGATACCTCTACCAGCACTGCCTATTTGGAAATCAACAGCCTCAGAAGTGATGACACGGCTATATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 259. CD33 AH3 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFK F12QHL GRVTMSSDTSTSTAYLEINSLRSDDTAIYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 260. CD33 AH3 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAAAAGCCTGGAGAGTCAGTCAAGGTCTCCTG F12QHL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAGGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGTCTTCGGATACCTCTACCAGCACTGCCTATTTGGAAATCAACAGCCTCAGAAGTGATGACACGGCTATATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 261. CD33 AH3 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVRQAPGQGLEWMGWINTYTGEPTYADDFK I2C HLGRVTMSSDTSTSTAYLEINSLRSDDTAIYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 262. CD33 AH3 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAAAAGCCTGGAGAGTCAGTCAAGGTCTCCTG I2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAGGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGTCTTCGGATACCTCTACCAGCACTGCCTATTTGGAAATCAACAGCCTCAGAAGTGATGACACGGCTATATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 263. CD33 AF5 HL x artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK H2C HLGRVTMTSDTSTSTAYLELHNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 264. CD33 AF5 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCGTCAGTCAAGGTCTCCTG H2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATTTGGAACTCCACAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 265. CD33 AF5 HL x artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK F12QHL GRVTMTSDTSTSTAYLELHNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 266. CD33 AF5 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCGTCAGTCAAGGTCTCCTG F12QHL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATTTGGAACTCCACAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 267. CD33 AF5 HL x artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK I2C HLGRVTMTSDTSTSTAYLELHNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 268. CD33 AF5 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCGTCAGTCAAGGTCTCCTG I2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATTTGGAACTCCACAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 269. CD33 AC8 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK H2C HLGRVTMTTDTSTSTAYMEIRNLRNDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 270. CD33 AC8 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG H2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCAGAAATGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGCTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 271. CD33 AC8 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK F12QHL GRVTMTTDTSTSTAYMEIRNLRNDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 272. CD33 AC8 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG F12QHL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCAGAAATGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 273. CD33 AC8 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK I2C HLGRVTMTTDTSTSTAYMEIRNLRNDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 274. CD33 AC8 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG I2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCAGAAATGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 275. CD33 AH11 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK H2C HLGRVTMTSDTSTSTAYMEISSLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 276. CD33 AH11 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG H2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATATGGAAATCAGCAGCCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 277. CD33 AH11 HL × artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK F12QHL GRVTMTSDTSTSTAYMEISSLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 278. CD33 AH11 HL ×artificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG F12QHL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATATGGAAATCAGCAGCCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 279. CD33 AH11 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFK I2C HLGRVTMTSDTSTSTAYMEISSLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 280. CD33 AH11 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG I2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATATGGAAATCAGCAGCCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 281. CD33 B3 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGETNYADKFQ H2C HLGRVTFTSDTSTSTAYMELRNLKSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSMTVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 282. CD33 B3 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG H2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGACAAACTATGCTGATAAGTTCCAGGGACGCGTTACCTTCACTTCGGATACCTCTACCAGCACTGCCTATATGGAACTCCGCAACCTCAAAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCATGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGACATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 283. CD33 B3 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGETNYADKFQ F12QHL GRVTFTSDTSTSTAYMELRNLKSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSMTVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 284. CD33 B3 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG F12QHL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGACAAACTATGCTGATAAGTTCCAGGGACGCGTTACCTTCACTTCGGATACCTCTACCAGCACTGCCTATATGGAACTCCGCAACCTCAAAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCATGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGACATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 285. CD33 B3 artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGETNYADKFQ HL xI2C HL GRVTFTSDTSTSTAYMELRNLKSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSMTVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 286. CD33 B3artificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG HL xI2C HL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGACAAACTATGCTGATAAGTTCCAGGGACGCGTTACCTTCACTTCGGATACCTCTACCAGCACTGCCTATATGGAACTCCGCAACCTCAAAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCATGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGACATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 287. CD33 F2 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGETNYADKFQ H2C HLGRVTFTSDTSTSTAYMELRNLKSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLSVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 288. CD33 F2 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG H2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGACAAACTATGCTGATAAGTTCCAGGGACGCGTTACCTTCACTTCGGATACCTCTACCAGCACTGCCTATATGGAACTCCGCAACCTCAAAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGTCTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 289. CD33 F2 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGETNYADKFQ F12QHL GRVTFTSDTSTSTAYMELRNLKSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLSVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 290. CD33 F2 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG F12QHL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGACAAACTATGCTGATAAGTTCCAGGGACGCGTTACCTTCACTTCGGATACCTCTACCAGCACTGCCTATATGGAACTCCGCAACCTCAAAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGTCTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 291. CD33 F2 artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGETNYADKFQ HL xI2C HL GRVTFTSDTSTSTAYMELRNLKSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLSVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 292. CD33 F2artificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG HL xI2C HL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGACAAACTATGCTGATAAGTTCCAGGGACGCGTTACCTTCACTTCGGATACCTCTACCAGCACTGCCTATATGGAACTCCGCAACCTCAAAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGTCTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 293. CD33 B10 artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQ HL xH2C HL GRVTMTTDTSTSTAYMEIRNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSNNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDGLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 294. CD33 B10artificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGTGAGTCAGTCAAGGTCTCCTG HL xH2C HL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACCTATGCTGATAAGTTCCAGGGACGCGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAACAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGTTCTGGGACAGATTTCACTCTCACTATTGACGGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 295. CD33 B10 artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQ HL xF12Q GRVTMTTDTSTSTAYMEIRNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsgggHL gsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSNNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDGLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 296. CD33 B10artificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGTGAGTCAGTCAAGGTCTCCTG HL xF12Q CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGHL GTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACCTATGCTGATAAGTTCCAGGGACGCGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAACAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGTTCTGGGACAGATTTCACTCTCACTATTGACGGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 297. CD33 B10 artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQ HL xI2C GRVTMTTDTSTSTAYMEIRNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSggggsggg HLgsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSNNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDGLQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 298. CD33 B10artificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGTGAGTCAGTCAAGGTCTCCTG HL xI2C CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGG HLGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACCTATGCTGATAAGTTCCAGGGACGCGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAACAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGTTCTGGGACAGATTTCACTCTCACTATTGACGGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 299. CD33 E11 artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQ HL xH2C GRVTMTTDTSTSTAYMEIRNLGGDDTAVYYCARWSWSDGYYVYFDYWGQGTSVTVSSggggsggg HLgsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSPQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 300. CD33 E11artificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG HL xH2C CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGG HLGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACCTATGCTGATAAGTTCCAGGGACGCGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCGGAGGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTTCGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCCGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 301. CD33 E11 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQ F12QHL GRVTMTTDTSTSTAYMEIRNLGGDDTAVYYCARWSWSDGYYVYFDYWGQGTSVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSPQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 302. CD33 E11 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG F12QHL CAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACCTATGCTGATAAGTTCCAGGGACGCGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCGGAGGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTTCGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCCGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 303. CD33 E11 HL x artificial aaQVQLVQSGAEVKKPGESVKVSCKASGYTFTNYGMNWVKQAPGQGLEWMGWINTYTGEPTYADKFQ I2C HLGRVTMTTDTSTSTAYMEIRNLGGDDTAVYYCARWSWSDGYYVYFDYWGQGTSVTVSSggggsggggsggggsDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSTNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSPQPEDSATYYCQQSAHFPITFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 304. CD33 E11 HL xartificial ntCAGGTGCAGCTGGTGCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGAGTCAGTCAAGGTCTCCTG I2C HLCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAGAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACCTATGCTGATAAGTTCCAGGGACGCGTTACCATGACTACGGATACCTCTACCAGCACTGCCTATATGGAAATCCGCAACCTCGGAGGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTTCGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCACGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCCGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAATCCGGAGGTGGTGGCTCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 305. CD33 human ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCGATCCAAATTTCTGGCTGCAAGTGCAGGAGTCAGTGACGGTACAGGAGGGTTTGTGCGTCCTCGTGCCCTGCACTTTCTTCCATCCCATACCCTACTACGACAAGAACTCCCCAGTTCATGGTTACTGGTTCCGGGAAGGAGCCATTATATCCGGGGACTCTCCAGTGGCCACAAACAAGCTAGATCAAGAAGTACAGGAGGAGACTCAGGGCAGATTCCGCCTCCTTGGGGATCCCAGTAGGAACAACTGCTCCCTGAGCATCGTAGACGCCAGGAGGAGGGATAATGGTTCATACTTCTTTCGGATGGAGAGAGGAAGTACCAAATACAGTTACAAATCTCCCCAGCTCTCTGTGCATGTGACAGACTTGACCCACAGGCCCAAAATCCTCATCCCTGGCACTCTAGAACCCGGCCACTCCAAAAACCTGACCTGCTCTGTGTCCTGGGCCTGTGAGCAGGGAACACCCCCGATCTTCTCCTGGTTGTCAGCTGCCCCCACCTCCCTGGGCCCCAGGACTACTCACTCCTCGGTGCTCATAATCACCCCACGGCCCCAGGACCACGGCACCAACCTGACCTGTCAGGTGAAGTTCGCTGGAGCTGGTGTGACTACGGAGAGAACCATCCAGCTCAACGTCACCTATGTTCCACAGAACCCAACAACTGGTATCTTTCCAGGAGATGGCTCAGGGAAACAAGAGACCAGAGCAGGAGTGGTTCATGGGGCCATTGGAGGAGCTGGTGTTACAGCCCTGCTCGCTCTTTGTCTCTGCCTCATCTTCTTCATAGTGAAGACCCACAGGAGGAAAGCAGCCAGGACAGCAGTGGGCAGGAATGACACCCACCCTACCACAGGGTCAGCCTCCCCGAAACACCAGAAGAAGTCCAAGTTACATGGCCCCACTGAAACCTCAAGCTGTTCAGGTGCCGCCCCTACTGTGGAGATGGATGAGGAGCTGCATTATGCTTCCCTCAACTTTCATGGGATGAATCCTTCCAAGGACACCTCCACCGAATACTCAGAGGTCAGGACCCAGTCCGGGCATCATCACCATCATCATTGA 306. CD33 human aaMGWSCIILFLVATATGVHSDPNFWLQVQESVTVQEGLCVLVPCTFFHPIPYYDKNSPVHGYWFREGAIISGDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMERGSTKYSYKSPQLSVHVTDLTHRPKILIPGTLEPGHSKNLTCSVSWACEQGTPPIFSWLSAAPTSLGPRTTHSSVLIITPRPQDHGTNLTCQVKFAGAGVTTERTIQLNVTYVPQNPTTGIFPGDGSGKQETRAGVVHGAIGGAGVTALLALCLCLIFFIVKTHRRKAARTAVGRNDTHPTTGSASPKHQKKSKLHGPTETSSCSGAAPTVEMDEELHYASLNFHGMNPSKDTSTEYSEVRTQSGHHHHHH 307. CD33 macaque ntATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCCCTGGCTATGGATCCAAGAGTCAGGCTGGAAGTGCAGGAGTCAGTGACAGTACAGGAGGGTTTGTGCGTCCTTGTGCCCTGCACTTTCTTCCATCCCGTACCCTACCACACCAGGAATTCCCCAGTTCATGGTTACTGGTTCCGGGAAGGAGCCATTGTATCCTTGGACTCTCCAGTGGCCACAAACAAGCTAGATCAAGAAGTACAGGAGGAGACCCAGGGCCGATTCCGCCTCCTTGGGGATCCCAGTAGGAACAACTGCTCCCTGAGCATCGTAGATGCCAGGAGGAGGGATAACGGTTCATACTTCTTTCGGATGGAGAAAGGAAGTACCAAATACAGTTACAAATCTACCCAGCTCTCTGTGCATGTGACAGACTTGACCCACAGGCCCCAAATCCTCATCCCTGGAGCCCTAGACCCTGACCACTCCAAAAACCTGACCTGCTCTGTGCCCTGGGCCTGTGAGCAGGGAACACCTCCAATCTTCTCCTGGATGTCAGCTGCCCCCACCTCCCTGGGCCTCAGGACCACTCACTCCTCGGTGCTCATAATCACCCCACGGCCCCAGGACCACGGCACCAACCTCACCTGTCAGGTGAAGTTCCCTGGAGCTGGCGTGACCACGGAGAGAACCATCCAGCTCAATGTCTCCTATGCTTCACAGAACCCAAGAACTGATATCTTTCTAGGAGACGGCTCAGGGAAACAAGGAGTGGTTCAGGGAGCCATCGGGGGAGCTGGTGTCACAGTCCTGCTCGCTCTTTGTCTCTGCCTCATCTTCTTCACAGTGAAGACTCACAGGAGGAAAGCAGCCAGGACAGCAGTGGGCAGGATCGACACCCACCCCGCCACAGGGCCAACATCCTCGAAACACCAGAAGAAGTCCAAGTTACATGGCGCCACTGAAACCTCAGGCTGTTCAGGTACCACCCTTACTGTGGAGATGGATGAGGAGCTGCACTACGCTTCCCTCAACTTTCATGGGATGAATCCTTCTGAGGACACCTCCACCGAATACTCAGAGGTCAGGACCCAGTGA 308. CD33 macaque aaMPLLLLLPLLWAGALAMDPRVRLEVQESVTVQEGLCVLVPCTFFHPVPYHTRNSPVHGYWFREGAIVSLDSPVATNKLDQEVQEETQGRFRLLGDPSRNNCSLSIVDARRRDNGSYFFRMEKGSTKYSYKSTQLSVHVTDLTHRPQILIPGALDPDHSKNLTCSVPWACEQGTPPIFSWMSAAPTSLGLRTTHSSVLIITPRPQDHGTNLTCQVKFPGAGVTTERTIQLNVSYASQNPRTDIFLGDGSGKQGVVQGAIGGAGVTVLLALCLCLIFFTVKTHRRKAARTAVGRIDTHPATGPTSSKHQKKSKLHGATETSGCSGTTLTVEMDEELHYASLNFHGMNPSEDTSTEYSEVRTQ 309. 1-27 CD3-Fc + artificial ntATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTACACTCCCAAGATGG LeaderTAATGAAGAAATGGGTGGTATTACACAGACACCATATAAAGTCTCCATCTCTGGAACCACAGTAATATTGACATCCGGAGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGTAAATAG 310.1-27 CD3-Fc + artificial aaMGWSCIILFLVATATGVHSQDGNEEMGGITQTPYKVSISGTTVILTSGEPKSCDKTHTCPPCPAP LeaderELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 311. CD33 UD H2C artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS HL xAF5 HL VKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVLSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFKGRVTMTSDTSTSTAYLELHNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIK 312. CD33 UD H2Cartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATG HL xAF5 HL TGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTATCCGGAGGTGGTGGCTCCCAGGTGCAGCTGGTCCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCGTCAGTCAAGGTCTCCTGCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATTTGGAACTCCACAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAA 313. CD33 UD F12Q artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS HL xAF5 HL VKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFKGRVTMTSDTSTSTAYLELHNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIK 314. CD33 UDartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATG F12QHLx AF5 TGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGHL GTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTATCCGGAGGTGGTGGCTCCCAGGTGCAGCTGGTCCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCGTCAGTCAAGGTCTCCTGCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATTTGGAACTCCACAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAA 315. CD33 UD I2C artificial aaEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADS HL xAF5 HL VKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVLSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGMNWVKQAPGQGLKWMGWINTYTGEPTYADDFKGRVTMTSDTSTSTAYLELHNLRSDDTAVYYCARWSWSDGYYVYFDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLTVSLGERTTINCKSSQSVLDSSKNKNSLAWYQQKPGQPPKLLLSWASTRESGIPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQSAHFPITFGQGTRLEIK 316. CD33 UD I2Cartificial ntGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATG HL xAF5 HL TGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTATCCGGAGGTGGTGGCTCCCAGGTGCAGCTGGTCCAGTCTGGAGCTGAGGTGAAGAAGCCTGGAGCGTCAGTCAAGGTCTCCTGCAAGGCTAGCGGGTATACCTTCACAAACTATGGAATGAACTGGGTGAAGCAGGCTCCAGGACAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACACTGGAGAGCCAACATATGCTGATGACTTCAAGGGACGGGTTACCATGACTTCGGATACCTCTACCAGCACTGCCTATTTGGAACTCCACAACCTCAGAAGTGATGACACGGCTGTATATTACTGTGCGCGCTGGAGTTGGAGTGATGGTTACTACGTTTACTTTGACTACTGGGGCCAAGGCACTACGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAGACAGCTCCAAGAATAAGAACTCCTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAATTACTCCTTTCCTGGGCATCTACGCGGGAATCCGGGATCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACTATTGACAGCCTGCAGCCTGAAGATTCTGCAACTTACTATTGTCAACAGTCTGCCCACTTCCCGATCACCTTTGGCCAAGGGACACGACTGGAGATTAAA 317. MCSP-A9 HL x artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYPFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ H2C HLGRVTITADESTSTAYMELSRLRSDDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFNTPFAFGQGTKLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 318. MCSP-A9 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG H2C HLCAAGGCTTCTGGATACCCCTTCACCGGCTACTACATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTCTTATCCAGCTCCAACAATAAGAACTACTTAAATTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCGGTTTATTACTGTCAACAACATTTTAATACTCCGTTCGCTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA319. MCSP-A9 HL x artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYPFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ F12QHL GRVTITADESTSTAYMELSRLRSDDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFNTPFAFGQGTKLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 320. MCSP-A9 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG F12QHL CAAGGCTTCTGGATACCCCTTCACCGGCTACTACATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTCTTATCCAGCTCCAACAATAAGAACTACTTAAATTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCGGTTTATTACTGTCAACAACATTTTAATACTCCGTTCGCTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA321. MCSP-A9 HL x artificial aaQVQLVQSGAEVKKPGASVKVSCKASGYPFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQ I2C HLGRVTITADESTSTAYMELSRLRSDDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFNTPFAFGQGTKLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 322. MCSP-A9 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTG I2C HLCAAGGCTTCTGGATACCCCTTCACCGGCTACTACATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGAGTCACGATTACCGCGGACGAATCCACGAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTCTTATCCAGCTCCAACAATAAGAACTACTTAAATTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCGGTTTATTACTGTCAACAACATTTTAATACTCCGTTCGCTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA323. MCSP-C8 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISGLQAEDVAVYYCQQHYSTPFTFGPGTKVDIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 324. MCSP-C8 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGTGGCCTGCAGGCTGAAGATGTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCCTGGGACCAAAGTGGATATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA325. MCSP-B8 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLTVSPGERATINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQHYSTPFTFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 326. MCSP-B8 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGACTGTGTCTCCGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCGATTCCCTGCAGCCTGAAGATAGTGCAACTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCAGGGGACCAGACTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA327. MCSP-B7 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLTVSLGERTTINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIDSLQPEDIATYYCQQHYSTPFTFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 328. MCSP-B7 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGACCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCGATTCCCTGCAGCCTGAAGATATTGCAACTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCAGGGGACCAGACTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA329. MCSP-G8 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLTVSLGERATINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQHYSTPFTFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 330. MCSP-G8 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCGATTCCCTGCAGCCTGAAGATAGTGCAACTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCAGGGGACCAGACTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA331. MCSP-D5 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIDSLQAEDSATYYCQQHYSTPFTFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 332. MCSP-D5 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCGATTCCCTGCAGGCTGAAGATAGTGCAACTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCAGGGGACCAGACTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA333. MCSP-F7 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIDVLQPEDIATYYCQQHYSTPFTFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 334. MCSP-F7 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCGATGTCCTGCAGCCTGAAGATATTGCAACTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCAGGGGACCAGACTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA335. MCSP-G5 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGDRATINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIDSLQPEDSATYYCQQHYSTPFTFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 336. MCSP-G5 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGACAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCGATTCCCTGCAGCCTGAGGATAGTGCAACTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCAGGGGACCAGACTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA337. MCSP-F8 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLTVSLGERATINCKSSQSVLNSKNNRNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTIDSLQAEDSAIYYCQQHYSTPFTFGQGTRLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 338. MCSP-F8 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACCCAGTCTCCAGACTCCCTGACTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTTTTAAACAGCAAGAACAATAGGAACTACTTAGCTTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGCTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCGATTCCCTGCAGGCTGAAGATAGTGCAATTTATTACTGTCAGCAACATTATAGTACTCCATTCACTTTTGGCCAGGGGACCAGACTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA339. MCSP-G10 HL x artificial aaQVQLVQSGAEVKRPGASMKVSCKASGYTFTNYYIHWVRQAPGQGLEWMGWINPNSGATNYAQKFQ I2C HLGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAKSWVSWFASWGQGTLVTVSSGGGGSGGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSVLSSSNNKNYLNWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHFNTPFAFGQGTKLEIKSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 340. MCSP-G10 HL xartificial ntCAGGTGCAGCTGGTCCAGTCTGGGGCTGAGGTGAAGAGGCCTGGGGCCTCAATGAAGGTCTCCTG I2C HLCAAGGCTTCTGGGTACACCTTCACCAACTACTATATACACTGGGTGCGACAGGCCCCTGGACAAGGTCTTGAGTGGATGGGTTGGATCAACCCTAACAGTGGTGCCACAAACTATGCACAGAAGTTCCAGGGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAAATCCTGGGTCTCCTGGTTTGCTTCCTGGGGTCAAGGAACCTTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTGACATCGTGATGACACAGTCTCCAGACTCCCTGGCTGTGTCTCTGGGCGAGAGGGCCACCATCAACTGCAAGTCCAGCCAGAGTGTCTTATCCAGCTCCAACAATAAGAACTACTTAAATTGGTACCAGCAGAAACCAGGACAGCCTCCTAAGTTGCTCATTTACTGGGCATCTACCCGGGAATCCGGGGTCCCTGACCGATTCAGTGGCAGCGGGTCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGGCTGAAGATGTGGCGGTTTATTACTGTCAACAACATTTTAATACTCCGTTCGCTTTTGGCCAGGGGACCAAGCTGGAGATCAAATCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTG TCCTA341. Human human aa QDGNEEMG CD3ε 1-8 (N-terminus) 342. Saimiri Saimiriaa QDGNEEIG sciureus CD3ε sciureus 1-8 (N-terminus) 343. Thioate-artificial nt TCCATGACGTTCCTGATGCT modified CpG- Oligo- nucleotide 344.MVH1 artificial nt (GC) AGGTGCAGCTCGAGGAGTCAGGACCT 345. MVH2 artificialnt GAGGTCCAGCTCGAGCAGTCTGGACCT 346. MVH3 artificial ntCAGGTCCAACTCGAGCAGCCTGGGGCT 347. MVH4 artificial ntGAGGTTCAGCTCGAGCAGTCTGGGGCA 348. MVH5 artificial ntGA(AG)GTGAAGCTCGAGGAGTCTGGAGGA 349. MVH6 artificial ntGAGGTGAAGCTTCTCGAGTCTGGAGGT 350. MVH7 artificial ntGAAGTGAAGCTCGAGGAGTCTGGGGGA 351. MVH8 artificial ntGAGGTTCAGCTCGAGCAGTCTGGAGCT 352. MuVHBstEII artificial ntTGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG 353. MUVK1 artificial ntCCAGTTCCGAGCTCGTTGTGACTCAGGAATCT 354. MUVK2 artificial ntCCAGTTCCGAGCTCGTGTTGACGCAGCCGCCC 355. MUVK3 artificial ntCCAGTTCCGAGCTCGTGCTCACCCAGTCTCCA 356. MUVK4 artificial ntCCAGTTCCGAGCTCCAGATGACCCAGTCTCCA 357. MUVK5 artificial ntCCAGATGTGAGCTCGTGATGACCCAGACTCCA 358. MUVK6 artificial ntCCAGATGTGAGCTCGTCATGACCCAGTCTCCA 359. MUVK7 artificial ntCCAGTTCCGAGCTCGTGATGACACAGTCTCCA 360. MuVkHindIII/ artificial ntTGGTGCACTAGTCGTACGTTTGATCTCAAGCTTGGTCCC BsiW1 361. forward primerartificial nt GATCTGGTCTACACCATCGAGC 362. reverse primer artificial ntGGAGCTGCTGCTGGCTCAGTGAGG 363. forward primer artificial ntTTCCAGCTGAGCATGTCTGATGG 364. reverse primer artificial ntCGATCAGCATCTGGGCCCAGG 365. forward primer artificial ntGTGGAGCAGTTCACTCAGCAGGACC 366. reverse primer artificial ntGCCTTCACACCCAGTACTGGCC 367. forward primer artificial ntTCCCGTACGAGATCTGGATCCCAATTGGATGGCGGACTCGTGCTGTTCTCACACAGAGG 368. reverseprimer artificial nt AGTGGGTCGACTCACACCCAGTACTGGCCATTCTTAAGGGCAGGG 369.forward primer artificial ntGAGGAATTCACCATGCCGCTGCTGCTACTGCTGCCCCTGCTGTGGGCAGGGGCCCTGGCTATGG 370.reverse primer artificial nt GATTTGTAACTGTATTTGGTACTTCC 371. forwardprimer artificial nt ATTCCGCCTCCTTGGGGATCC 372. reverse primerartificial nt GCATAGGAGACATTGAGCTGGATGG 373. forward primer artificialnt GCACCAACCTGACCTGTCAGG 374. reverse primer artificial ntAGTGGGTCGACTCACTGGGTCCTGACCTCTGAGTATTCG 375. V EGFR 3D-E8 artificial aaEVQLLESGGGLVQAGGSLRLSCAASGRTFSTYTMAWFRQAPGKEREFVQGISRSDGGTYDADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAAASVKLVYVNPNRYSYWGQGTQVTVSS 376. V EGFR3D-E8 artificial aa TYTMA CDR1 377. V EGFR 3D-E8 artificial aaGISRSDGGTYDADSVKG CDR2 378. V EGFR 3D-E8 artificial aa ASVKLVYVNPNRYSYCDR3 379. EGFR 3D-E8 artificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTCTGGTTCAGGCGGGTGGCAGCCTGCGTCTGAGCTGTGCGGCGAGCGGCCGTACCTTTAGCACCTATACCATGGCGTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGCAGGGCATTAGCCGTAGCGATGGCGGCACCTATGATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTTTTGCGCGGCAGCGAGCGTGAAACTGGTGTATGTGAATCCGAACCGTTATAGCTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCC 380. EGFR 3D-E8 xartificial aaEVQLLESGGGLVQAGGSLRLSCAASGRTFSTYTMAWFRQAPGKEREFVQGISRSDGGTYDADSVK I2C HLGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAAASVKLVYVNPNRYSYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 381. EGFR 3D-E8 xartificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTCTGGTTCAGGCGGGTGGCAGCCTGCGTCTGAGCTG I2C HLTGCGGCGAGCGGCCGTACCTTTAGCACCTATACCATGGCGTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGCAGGGCATTAGCCGTAGCGATGGCGGCACCTATGATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTTTTGCGCGGCAGCGAGCGTGAAACTGGTGTATGTGAATCCGAACCGTTATAGCTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 382. EGFR 3D-E8 x artificial aaEVQLLESGGGLVQAGGSLRLSCAASGRTFSTYTMAWFRQAPGKEREFVQGISRSDGGTYDADSVK H2C HLGRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAAASVKLVYVNPNRYSYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 383. EGFR 3D-E8 xartificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTCTGGTTCAGGCGGGTGGCAGCCTGCGTCTGAGCTG H2C HLTGCGGCGAGCGGCCGTACCTTTAGCACCTATACCATGGCGTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGCAGGGCATTAGCCGTAGCGATGGCGGCACCTATGATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTTTTGCGCGGCAGCGAGCGTGAAACTGGTGTATGTGAATCCGAACCGTTATAGCTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 384. EGFR 3D-E8 x artificial aaEVQLLESGGGLVQAGGSLRLSCAASGRTFSTYTMAWFRQAPGKEREFVQGISRSDGGTYDADSVK F12QHL GRFTISRDNAKNTVYLQMNSLKPEDTAVYFCAAASVKLVYVNPNRYSYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 385. EGFR 3D-E8 xartificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTCTGGTTCAGGCGGGTGGCAGCCTGCGTCTGAGCTG F12QHL TGCGGCGAGCGGCCGTACCTTTAGCACCTATACCATGGCGTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGCAGGGCATTAGCCGTAGCGATGGCGGCACCTATGATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGTATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGGTGTATTTTTGCGCGGCAGCGAGCGTGAAACTGGTGTATGTGAATCCGAACCGTTATAGCTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 386. V EGFR 3D-D12 artificial aaEVQLLESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSS 387. V EGFR3D-D12 artificial aa SYGMG CDR1 388. V EGFR 3D-D12 artificial aaGISWRGDSTGYADSVKG CDR2 389. V EGFR 3D-D12 artificial aa AAGSAWYGTLYEYDYCDR3 390. V EGFR 3D-D12 artificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTAGCGTGCAGACCGGCGGTAGCCTGCGTCTGACCTGTGCGGCGAGCGGTCGTACCAGCCGTAGCTATGGCATGGGCTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGAGCGGCATTAGCTGGCGTGGCGATAGCACCGGCTATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGGATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGATTTATTATTGCGCGGCAGCGGCGGGTAGCGCGTGGTATGGCACCCTGTATGAATATGATTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCC 391. V EGFR 3D-D12artificial aaEVQLLESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVK x I2CHL GRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 392. V EGFR 3D-D12artificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTAGCGTGCAGACCGGCGGTAGCCTGCGTCTGACCTG x I2CHL TGCGGCGAGCGGTCGTACCAGCCGTAGCTATGGCATGGGCTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGAGCGGCATTAGCTGGCGTGGCGATAGCACCGGCTATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGGATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGATTTATTATTGCGCGGCAGCGGCGGGTAGCGCGTGGTATGGCACCCTGTATGAATATGATTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 393. V EGFR 3D-D12 artificial aaEVQLLESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVK x H2CHL GRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNKYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYISYWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGYYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCALWYSNRWVFGGGTKLTVL 394. V EGFR 3D-D12artificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTAGCGTGCAGACCGGCGGTAGCCTGCGTCTGACCTG x H2CHL TGCGGCGAGCGGTCGTACCAGCCGTAGCTATGGCATGGGCTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGAGCGGCATTAGCTGGCGTGGCGATAGCACCGGCTATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGGATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGATTTATTATTGCGCGGCAGCGGCGGGTAGCGCGTGGTATGGCACCCTGTATGAATATGATTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAAGTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGACAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACATATCCTACTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCTACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGCTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 395. V EGFR 3D-D12 artificial aaEVQLLESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVK x F12QHL GRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSGGGGSEVQLVESGGGLVQPGGSLKLSCAASGFTFNSYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKGRFTISRDDSKNTAYLQMNNLKTEDTAVYYCVRHGNFGNSYVSWWAYWGQGTLVTVSSGGGGSGGGGSGGGGSQTVVTQEPSLTVSPGGTVTLTCGSSTGAVTSGNYPNWVQQKPGQAPRGLIGGTKFLAPGTPARFSGSLLGGKAALTLSGVQPEDEAEYYCVLWYSNRWVFGGGTKLTVL 396. V EGFR 3D-D12artificial ntGAGGTGCAGCTGCTCGAGAGCGGTGGTGGTAGCGTGCAGACCGGCGGTAGCCTGCGTCTGACCTG x F12QHL TGCGGCGAGCGGTCGTACCAGCCGTAGCTATGGCATGGGCTGGTTTCGTCAGGCACCGGGCAAAGAACGTGAATTTGTGAGCGGCATTAGCTGGCGTGGCGATAGCACCGGCTATGCGGATAGCGTGAAAGGCCGTTTTACCATTAGCCGTGATAACGCGAAAAACACCGTGGATCTGCAGATGAACAGCCTGAAACCGGAAGATACCGCGATTTATTATTGCGCGGCAGCGGCGGGTAGCGCGTGGTATGGCACCCTGTATGAATATGATTATTGGGGCCAGGGCACCCAGGTGACCGTTAGCTCCGGAGGTGGTGGATCCGAGGTGCAGCTGGTCGAGTCTGGAGGAGGATTGGTGCAGCCTGGAGGGTCATTGAAACTCTCATGTGCAGCCTCTGGATTCACCTTCAATAGCTACGCCATGAACTGGGTCCGCCAGGCTCCAGGAAAGGGTTTGGAATGGGTTGCTCGCATAAGAAGTAAATATAATAATTATGCAACATATTATGCCGATTCAGTGAAAGGCAGGTTCACCATCTCCAGAGATGATTCAAAAAACACTGCCTATCTACAAATGAACAACTTGAAAACTGAGGACACTGCCGTGTACTACTGTGTGAGACATGGGAACTTCGGTAATAGCTACGTTTCCTGGTGGGCTTACTGGGGCCAAGGGACTCTGGTCACCGTCTCCTCAGGTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGACTGTTGTGACTCAGGAACCTTCACTCACCGTATCACCTGGTGGAACAGTCACACTCACTTGTGGCTCCTCGACTGGGGCTGTTACATCTGGCAACTACCCAAACTGGGTCCAACAAAAACCAGGTCAGGCACCCCGTGGTCTAATAGGTGGGACTAAGTTCCTCGCCCCCGGTACTCCTGCCAGATTCTCAGGCTCCCTGCTTGGAGGCAAGGCTGCCCTCACCCTCTCAGGGGTACAGCCAGAGGATGAGGCAGAATATTACTGTGTTCTATGGTACAGCAACCGCTGGGTGTTCGGTGGAGGAACCAAACTGACTGTCCTA 397. V CD3 3D-H11 artificial aaEVQLLEEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKWLEWVSDISWNGGSTYYADSVKGRFTISRDNAENTLYLQMNSLKPDDTAVYYCAKMGEGGWGANDYWGQGTQVTVSS 398. V CD33D-H11 artificial aa DYGMS CDR1 399. V CD3 3D-H11 artificial aaDISWNGGSTYYADSVKG CDR2 400. V CD3 3D-H11 artificial aa MGEGGWGANDY CDR3401. V CD3 3D-H11 artificial ntGAGGTGCAGCTGCTCGAGGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTTGATGATTATGGCATGAGCTGGGTCCGACAGGCTCCAGGGAAGTGGCTGGAGTGGGTCTCAGATATTAGCTGGAATGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACGCCGAGAACACGCTGTATCTGCAAATGAACAGCCTGAAACCTGACGACACGGCCGTGTATTACTGTGCAAAAATGGGTGAAGGGGGATGGGGTGCAAATGACTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCC 402. 5′ EGFR AGartificial nt GGTCTAGAGCATGCGACCCTCCGGGACGGCCGGG XbaI 403. 3′ EGFR AGartificial nt TTTTAAGTCGACTCATGCTCCAATAAATTCACTGCT SalI 404. Forwardprimer artificial nt CGCTCTGCCCGGCGAGTCGGGC 405. Reverse primerartificial nt CCGTCTTCCTCCATCTCATAGC 406. Forward primer artificial ntACATCCGGAGGTGACAGATCACGGCTCGTGC 407. Reverse primer artificial ntCAGGATATCCGAACGATGTGGCGCCTTCGC 408. 5′-VHHa-XhoI artificial nt CTG ACGCTC GAG GAG GTG CAG CTG GTG GAG TCT GG 409. 5′-VHHb-XhoI artificial ntCTG ACG CTC GAG CAG GTR CAG CTG GTG GAG TCT GG 410. 5′-VHHc-XhoIartificial nt CTG ACG CTC GAG CAG GTA AAG CTG GAG GAG TCT GG 411.5′-VHHd-XhoI artificial nt CTG ACG CTC GAG GAT GTG CAG CTG GTG GAG TCTGG 412. 5′-VHHe-XhoI artificial nt CTG ACG CTC GAG GCC GTG CAG CTG GTGGAT TCT GG 413. 5′-VHHf-XhoI artificial nt ACG CTC GAG GCG GTG CAG CTGGTG GAG TCT GG- 414. 5′-VHH-LP-A- artificial nt CTG ACG CTC GAG GAG GTGCAG CTG CAG GCG TCT G XhoI 415. 5′-VHH-LP-B- artificial nt CTG ACG CTCGAG GAT GTS CAG CTG CAG GCG TCT G XhoI 416. 5′-VHH-LX-I- artificial ntCTG ACG CTC GAG CAG GTG CAG CTG GTG CAG TCT GG XhoI 417. 5′-VHH-LX-II-artificial nt CTG ACG CTC GAG CAG GTC ACC TTG AAG GAG TCT GG XhoI 418.5′-VHH-LX-III- artificial nt CTG ACG CTC GAG CAG GTG CAG CTG CAG GAG TCGGG XhoI 419. 5′-VHH-LG-1- artificial nt CTG ACG CTC GAG CTG CAG CAG TCTGGG GGA GG XhoI 420. 3′-VHHG2- artificial nt CTG ACG ACT AGT CGT ACG TTGGGG TAT CTT GGG TTC TG BsiWI-SpeI 421. 3′-VHHG3- artificial nt CTG ACGACT AGT CGT ACG TAC TTC ATT CGT TCC TGA VGA G BsiWI-SpeI 422.3′-VHH-LP-G2a- artificial nt CTG ACG ACT AGT CGT ACG TTG TGG TTT TGG TGTCTT GGG TTC BsiWI-SpeI 423. 3′-VHH-LP- artificial nt CTG ACG ACT AGT CGTACG TGA GGA GAC GGT GAC CTG GGT CC dirA-BsiWI- SpeI 424. 3′-VHH-LG-artificial nt CTG ACG ACT AGT CGT ACG GGT GAC CTG GGT CCC CTG GCdir1-BsiWI- SpeI 425. N-termina artificial aaQDGNEEMGGITQTPYKVSISGTTVILTC 1-27 + additional C at position 28 426.Flag-tag artificial aa YKDDDDK 427. Macaca Macaca aaQDGNEEMGSITQTPYQVSISGTTILTC fascicularis fascicularis CD3epsilon 1-27428. Macaca Macaca aa QDGNEEMGSITQTPYQVSISGTTVILT fascicularisfascicularis CD3epsilon 1-27 429. Macaca mulatta Macaca aaQDGNEEMGSITQTPYHVSISGTTVILT CD3epsilon mulatta 1-27

1. A bispecific single chain antibody molecule comprising a firstbinding domain consisting of one antibody variable domain which is anantigen-interactive site, capable of binding to an epitope of the humanand Callithrix jacchus, Sanguinis Oedipus or Saimiri sciureus CD3ε(epsilon) chain, wherein the epitope is part of an amino acid sequencecomprised in the group consisting of SEQ ID NOs. 2, 4, 6, or 8, andcomprises at least the amino acid sequence Gln-Asp-Gly-Asn-Glu (QDGNE) asecond binding domain capable of binding to an epitope of a human and anon-chimpanzee primate tumor target antigen.
 2. The bispecific singlechain antibody molecule of claim 1, wherein at least one of said firstor second binding domain is CDR-grafted, humanized or human.
 3. Thebispecific single chain antibody molecule of claim 1, wherein at leastone of said first or second binding domain is a VHH domain.
 4. Thebispecific single chain antibody molecule of claim 1, wherein theantibody variable domain of the first binding domain comprises CDR 1 ofSEQ ID NO: 398, CDR 2 of SEQ ID NO. 399 and CDR 3 of SEQ ID NO.
 400. 5.The bispecific single chain antibody molecule of claim 4, wherein thefirst binding domain comprises an antibody variable domain as shown inSEQ ID NO. 397 or an amino acid sequence at least 80%, more preferred atleast 90% or 95% identical, most preferred at least 96% identical to theamino acid sequence of SEQ ID NO.
 397. 6. The bispecific single chainantibody molecule of claim 1 wherein the second binding domain comprisesone antibody variable domain or two antibody variable domains.
 7. Thebispecific single chain antibody molecule of claim 1, wherein the tumortarget antigen is EGFR, CD44v6 or CD30.
 8. A bispecific single chainantibody molecule comprising a first binding domain capable of bindingto an epitope of human and non-chimpanzee primate CD3ε (epsilon) chain,wherein the epitope is part of an amino acid sequence comprised in thegroup consisting of SEQ ID NOs. 2, 4, 6, and 8, and a second bindingdomain consisting of one antibody variable domain capable of binding toan epitope of a human and a non-chimpanzee primate tumor target antigen.9. The bispecific single chain antibody molecule of claim 8, wherein atleast one of said first or second binding domain is CDR-grafted,humanized or human.
 10. The bispecific single chain antibody molecule ofclaim, wherein the first binding domain capable of binding to an epitopeof human and nonchimpanzee primate CD3ε chain comprises two antibodyvariable domains.
 11. The bispecific single chain antibody moleculeaccording to claim 10, wherein the first binding domain capable ofbinding to an epitope of human and nonchimpanzee primate CD3ε chaincomprises an amino acid sequence selected from the group consisting ofSEQ ID NOs: 23, 25, 41, 43, 59, 61, 77, 79, 95, 97, 113, 115, 131, 133,149, 151, 167, 169, 185 and
 187. 12. The bispecific single chainantibody molecule of claim 8, wherein the tumor target antigen is EGFR,CD44v6 or CD30.
 13. The bispecific single chain antibody molecule ofclaim 12, wherein the antibody variable domain of the second bindingdomain comprises CDR1-3 selected from the group consisting of: (a) CDR 1of SEQ ID NO: 376, CDR 2 of SEQ ID NO. 377 and CDR 3 of SEQ ID NO. 378;and (b) CDR 1 of SEQ ID NO: 387, CDR 2 of SEQ ID NO. 388 and CDR 3 ofSEQ ID NO.
 389. 14. The bispecific single chain antibody molecule ofclaim 13, wherein the second binding domain comprises an antibodyvariable domain as shown in SEQ ID NO. 375 or 386 or an amino acidsequence at least 80%, more preferred at least 90% or 95% identical,most preferred at least 96% identical to the amino acid sequence of SEQID NO. 375 or
 386. 15. The bispecific single chain antibody moleculeaccording to claim 14, wherein the bispecific single chain antibodymolecule comprises a sequence selected from: (a) an amino acid sequenceas depicted in any of SEQ ID NOs. 380, 382, 384, 391, 393 or 395; (b) anamino acid sequence encoded by a nucleic acid sequence as depicted inany of SEQ ID NOs: 381, 383, 385, 392, 394 or 396; and (c) an amino acidsequence at least 90% identical, more preferred at least 95% identical,most preferred at least 96% identical to the amino acid sequence of (a)or (b).
 16. The polypeptide as defined in claim 1, wherein the epitopeis part of an amino acid sequence comprised in the group consisting ofSEQ ID NOs:2, 4, 6 and 8 and comprises at least the amino acid sequenceGln-Asp-Gly-Asn-Glu.